α-amino esters of hydroxypropylthiazolidine carboxamide derivative and salt form, crystal polymorph thereof

ABSTRACT

The invention provides α-amino esters of a hydroxypropylthiazolidine carboxamide derivative, (2S)-3-([1,1′-biphenyl]-4-ylsulfonyl)-N-[(1S)-3-hydroxy-1-phenylpropyl]-1,3-thiazolidine-2-carboxamide, as well as salts and crystal polymorphs thereof, that can be used to inhibit prostaglandin F receptor. The invention further encompasses methods of treating disorders such as pre-term labor at the early gestational stage by the administration of these substances to a patient in need of treatment.

FIELD OF THE INVENTION

The invention relates to chemical compositions, such as compounds,salts, and crystal polymorphs, that are capable of binding andinhibiting the activity of prostaglandin F2α (PGF2α) receptor, as wellas methods of preventing pre-term labor at the early gestational stageby administration of these compositions to a patient in need oftreatment.

BACKGROUND OF THE INVENTION

Pre-term delivery represents a prevalent cause of perinatal mortality inthe developed world and occurs in approximately 7% to 10% of alldeliveries (Berkowitz et al. Epidemiol. Rev. 15:414-443 (1993)). Severemorbidity, especially respiratory distress syndrome, intraventricularhemorrhage, bronchopulmonary dysplasia and necrotizing enterocolitis,are far more common in pre-term than in term infants. Long-termimpairment, such as cerebral palsy, visual impairment and hearing loss,are also more common in pre-term infants. At present, pre-term birthremains a leading cause of infant mortality and morbidity in the UnitedStates, where, despite the significant improvements in obstetricalmedicine, the infant mortality rate is higher than in many otherindustrialized nations, causing costs exceeding $5 billion per year forneonatal intensive care of low birth-weight babies. The actual costsassociated with this care are even higher when taking into considerationthe healthcare provision of pre-term childbirth-related ailments, suchas respiratory distress syndrome, heart conditions, cerebral palsy,epilepsy, and severe learning disabilities.

During the past 40 years of clinical investigations, and despite the useof multiple therapeutic agents, the rate of pre-term birth has notdrastically declined. The prevention of pre-term labor is difficult andalthough tocolytic therapy remains the cornerstone of management ofpre-term labor, there is not universal agreement as to its value in thiscondition. The available tocolytic agents on their own do not prolonglabor for more than 48 hours, and the majority of these agents lackuterine selectivity and can thus cause potentially serious side effectsboth for the mother and the fetus.

Fundamentally, term and pre-term labor are similar processes in thatthey share a common physiological endpoint characterized by uterinecontractions, cervical dilatation, and activation of the fetalmembranes. The differences lie in the gestational age at which theseprocesses occur and the mechanisms by which they are activated. Termlabor is thought to result from physiological activation of the terminalpathway, whereas pre-term labor is a pathological conditioncharacterized by multiple etiologies in which one or more components ofthis pathway are aberrantly activated.

Uterine contractility is stimulated or inhibited by various receptors inmyometrial cells. It is hypothesized that activation of the myometriumresults from the coordinated expression of contraction-associatedproteins (CAPs), including actin, myosin, connexin-43, and the receptorsfor oxytocin and prostaglandins. In general, receptors that provokecalcium entry or calcium release from intracellular stores stimulatecontractility. However, receptors coupled to the production of cyclicnucleotides, such as cyclic adenosine monophosphate (cAMP) relax theuterus. For instance, oxytocin and prostaglandin F (FP) receptors arestimulatory, while β2 adrenoceptors and prostaglandin E2 receptorscoupled to cAMP formation are inhibitory.

In uterine tissues, prostaglandins E2 (PGE2) and F2α (PGF2α) have beenshown to induce cervical changes and elicit uterine contractility, twokey events in the physiology of labor and parturition. Activation of theFP receptor in the human myometrium by PGF2α results in the elevation ofintracellular calcium concentration, which, in turn, leads tocontraction of the uterine smooth cell muscle (Abramovitz et al. J.Biol. Chem. 269:2632-2636 (1994) and Senior, et al. Br. J. Pharmacol.108:501-506 (1993)). FP receptors are up-regulated in uterine tissuestowards term (Al-Matubsi et al. Biol. Reprod. 65:1029-1037 (2001)).Inhibitors of prostaglandin synthesis (such as indomethacin andnimesulide) have shown some tocolytic effect but are not devoid of sideeffects and their un-licensed use in the clinic has raised concernsregarding fetal safety (Norton et al. New Engl. J. Med. 329:1602-1067(1993) and Peruzzi et al. New Engl. J. Med. 354:1615 (1999)). Thereremains a need to develop therapeutics with myometrial selectivity thatpermit lasting inhibition of uterine contractions that lead to labor andthat prolong pregnancy to a stage where increased fetal maturationraises the chances of survival.

SUMMARY OF THE INVENTION

The invention encompasses alpha-amino esters of ahydroxypropylthiazolidine carboxamide derivative, as well as saltsthereof, that are capable of antagonizing the interaction betweenprostaglandin F2α (PGF2α) and the prostaglandin F receptor. Thesecompounds can be administered to a subject, such as a pregnant humanfemale subject, in order to treat or prevent preterm labor. Theinvention additionally provides methods of synthesizing these compounds,as well as methods for preparing crystal forms thereof.

In a first aspect, the invention provides a compound represented byformula (I),

(3S)-3-({[(2S)-3-(biphenyl-4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3-(4-fluorophenyl)propylL-valinate, or a pharmaceutically acceptable salt thereof. In someembodiments, the compound is represented by formula (III),(3S)-3-({[(2S)-3-(biphenyl-4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3-(4-fluorophenyl)propylL-valinate hydrochloride.

In some embodiments, the compound binds human prostaglandin F2α receptorwith an affinity of about 1 nM. Compounds of the invention demonstratethe ability to selectively bind prostaglandin F receptors, such asprostaglandin F2α, over other prostaglandin receptor subtypes. Forinstance, compounds of the invention exhibit an affinity forprostaglandin F2α receptor that is about 10-fold greater than thatobserved for prostaglandin E2 receptor. Additionally, compounds of theinvention exhibit an affinity for prostaglandin F2α receptor that isabout 100-fold or above (e.g., from about 100-fold to about 1,000-fold,such as about 100-fold, 110-fold, 120-fold, 130-fold, 140-fold,150-fold, 160-fold, 170-fold, 180-fold, 190-fold, 200-fold, 210-fold,220-fold, 230-fold, 240-fold, 250-fold, 260-fold, 270-fold, 280-fold,290-fold, 300-fold, 310-fold, 320-fold, 330-fold, 340-fold, 350-fold,360-fold, 370-fold, 380-fold, 390-fold, 400-fold, 410-fold, 420-fold,430-fold, 440-fold, 450-fold, 460-fold, 470-fold, 480-fold, 490-fold,500-fold, 510-fold, 520-fold, 530-fold, 540-fold, 550-fold, 560-fold,570-fold, 580-fold, 590-fold, 600-fold, 610-fold, 620-fold, 630-fold,640-fold, 650-fold, 660-fold, 670-fold, 680-fold, 690-fold, 700-fold,710-fold, 720-fold, 730-fold, 740-fold, 750-fold, 760-fold, 770-fold,780-fold, 790-fold, 800-fold, 810-fold, 820-fold, 830-fold, 840-fold,850-fold, 860-fold, 870-fold, 880-fold, 890-fold, 900-fold, 910-fold,920-fold, 930-fold, 940-fold, 950-fold, 960-fold, 970-fold, 980-fold,990-fold, 1,000-fold, or above) greater than other prostaglandinreceptor subtypes, such as prostaglandin E1, E3, E4, D1, D2, 11, and 12receptor subtypes. In some embodiments, the compound is soluble inaqueous solution at a concentration of from about 300 μg/mL to about 500μg/mL, such as at a concentration of about 380 μg/mL.

In some embodiments, the compound inhibits synthesis of inositoltriphosphate in a cell, such as a mammalian cell. In some embodiments,the mammalian cell is a human cell, such as a myometrial cell. In someembodiments, the myometrial cell is a uterine myocyte. In someembodiments, the compound induces a reduction in the amplitude ofuterine contractions in a subject following administration of thecompound to the subject. For instance, the compound may induce areduction of from about 40% to about 50% relative to a measurement ofthe amplitude of uterine contractions in the subject recorded prior tothe administration. In some embodiments, the compound exhibits a halflife in a subject of from about 1 to about 4 hours followingadministration of the compound to the subject. In some embodiments, thecompound reaches a maximum plasma concentration in a subject within fromabout 0.25 to about 2 hours following administration of the compound tothe subject.

In some embodiments, the subject is a mammal. In some embodiments, themammal is a human. In some embodiments, the mammal is a non-human, suchas canine or a rat. In some embodiments, the compound is administered tothe subject orally. In some embodiments, the compound is administered tothe subject intravenously.

In another aspect, the invention encompasses a compound represented byformula (III)

wherein the compound is in a crystalline state.

In some embodiments, the compound exhibits characteristic X-ray powderdiffraction peaks at about 7.0° 2θ, about 8.1° 2θ, about 10.0° 2θ, about20.1° 2θ, about 21.0° 2θ, and about 23.5° 20. In some embodiments, thecompound additionally exhibits X-ray powder diffraction peaks at about12.0° 2θ, about 13.1° 2θ, about 14.1° 2θ, about 16.4° 2θ, about 18.4°2θ, and about 29.5° 2θ. In some embodiments, the compound ischaracterized by an X-ray powder diffraction spectrum substantially asdepicted in any one of FIGS. 19, 22, 29, 45-49, and 54. For instance, insome embodiments, the compound is characterized by an X-ray powderdiffraction spectrum substantially as depicted in FIG. 49.

In some embodiments, the compound exhibits ¹H nuclear magnetic resonance(NMR) peaks centered at about 1.1 ppm, about 3.3 ppm, about 4.9 ppm,about 5.4 ppm, about 7.1 ppm, about 7.7 ppm, about 7.9 ppm, and about8.0 ppm. In some embodiments, the compound is characterized by a ¹H NMRspectrum substantially as depicted in FIG. 21.

In some embodiments, the compound exhibits an endotherm at from about145° C. to about 147° C. as measured by differential scanningcalorimetry. In some embodiments, the compound exhibits an additionalendotherm at about 214° C. as measured by differential scanningcalorimetry. In some embodiments, the compound is characterized by adifferential scanning calorimetry curve substantially as depicted inFIG. 20. In some embodiments, the compound exhibits an additionalendotherm at about 228° C. as measured by differential scanningcalorimetry. In some embodiments, the compound is characterized by adifferential scanning calorimetry curve substantially as depicted inFIG. 23.

In some embodiments, the compound exhibits a weight loss of from about0.2% to about 0.6% when heated from 25° C. to 100° C. as measured bythermogravimetric analysis. In some embodiments, the compound exhibits aweight loss of from about 2.5% to about 3.5% when heated from 100° C. to160° C. as measured by thermogravimetric analysis. In some embodiments,the compound exhibits a thermogravimetric analysis curve substantiallyas depicted in FIG. 24.

In an additional aspect, the invention provides a pharmaceuticalcomposition containing the compound of any of the above-describedaspects and optionally containing one or more excipients. In someembodiments, the compound has a purity of at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 99.9%, e.g., as ascertained by highpressure liquid chromatography (HPLC). In some embodiments, thepharmaceutical composition is formulated for oral administration to asubject. In some embodiments, the pharmaceutical composition is atablet, capsule, gel cap, powder, liquid solution, or liquid suspension.In some embodiments, the pharmaceutical composition is formulated forintravenous administration to a subject.

In some embodiments, the pharmaceutical composition comprises anoxytocin receptor antagonist, such as atosiban, retosiban, barusiban,epelsiban, and nolasiban, as well as derivatives thereof.

In another aspect, the invention provide a method of synthesizing acompound represented by formula (I)

or a pharmaceutically acceptable salt thereof by reacting a precursorrepresented by formula (IV)

with a precursor represented by formula (V)

to form an amino ester, wherein X is a protecting group. In someembodiments, the method includes deprotecting the amino ester. In someembodiments, the compound is represented by formula (III).

In some embodiments, the method includes reacting the amino ester with areagent capable of deprotecting the amino ester. In some embodiments,the protecting group is selected from the group consisting oftert-butoxycarbonyl, trityl, 4-monomethoxytrityl, 4-methyltrityl,3,5-dimethoxyphenylisopropoxycarbonyl, 2-(4-biphenyl)isopropoxycarbonyl, 2-nitrophenylsulfenyl, 9-fluorenylmethoxycarbonyl,2-(4-nitrophoneylsulfonyl)ethoxycarbonyl,(1,1-dioxobenzo[b]thiophene-2-yl)methoxycarbonyl,1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl,2,7-di-tert-butyl-9-fluorenylmethoxycarbonyl,2-fluoro-9-fluorenylmethoxycarbonyl,2-monoisooctyl-9-fluorenylmethoxycarbonyl,2,7-diisooctyl-9-fluorenylmethoxycarbonyl, tetrachlorophthaloyl,2-[phenyl(methyl)sulfonio]ethyloxycarbonyl tetrafluoroborate,ethanesulfonylethoxycarbonyl, 2-(4-sulfophenylsulfonyl)ethoxycarbonyl,benzyloxycarbonyl, allyloxycarbonyl, o-nitrobenzenesulfonyl,2,4-dinitrobenzenesulfonyl, benzothiazole-2-sulfonyl,2,2,2-trichloroethyloxycarbonyl, dithiasuccinoyl,p-nitrobenzyloxycarbonyl, an α-azidoacid, propargyloxycarbonyl,9-(4-bromophenyl)-9-fluorenyl, azidomethoxycarbonyl, hexafluoroacetone,2-chlorobenzyloxycarbonyl, trifluoroacetyl,2-(methylsulfonyl)ethoxycarbonyl, phenyldisulfanylethyloxycarbonyl, and2-pyridyldisulfanylethyloxycarbonyl.

In some embodiments, the reagent is selected from the group consistingof methanesulfonic acid, hydrochloric acid, trifluoroacetic acid, aceticacid, piperidine, 1,8-diazabicyclo[5.4.0]undec-7-ene, morpholine,hexamethyleneimine, ammonia, diethylamine, piperazine,tris(2-aminoethyl)amine, hydrazine, 1-methylpyrrolidine, sodium hydrogencarbonate, sodium hydroxide, barium hydroxide, sodium carbonate,molecular hydrogen, hydrobromic acid, boron tribromide,tetrakis(triphenylphosphine)palladium, thiophenol, β-mercaptoethanol,2-mercaptoacetic acid, aluminum amalgam, zinc, hypophosphorous acid,sodium borohydride, N-mercaptoacetamide, tin(II) chloride,trimethylphosphine, tributylphosphine, triphenylphosphine,benzyltriethylammonium tetrathiomolybdate, palladium(II) acetate,hydrofluoric acid, trimethylsilyl chloride, trimethylsilyltrifluoromethanesulfonate, and trifluoromethanesulfonic acid.

In some embodiments, the protecting group is tert-butoxycarbonyl and thereagent is selected from the group consisting of methanesulfonic acid,hydrochloric acid, and trifluoroacetic acid, such as methanesulfonicacid.

In some embodiments, the method includes exposing the amino ester toelectromagnetic radiation. In some embodiments, the protecting group isselected from the group consisting of o-nitrobenzyloxycarbonyl,4-nitroveratryloxycarbonyl, 2-(2-nitrophenyl)propyloxycarbonyl, and2-(3,4-methylenedioxy-6-nitrophenyl)propyloxycarbonyl. In someembodiments, the electromagnetic radiation is characterized by awavelength of from about 300 to about 400 nm.

In some embodiments, the method includes reacting the precursorrepresented by formula (IV) with the precursor represented by formula(V) and a diimide. In some embodiments, the diimide is selected from thegroup consisting of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide,N,N′-diisopropylcarbodiimide, and N,N′-diisopropylcarbodiimide. In someembodiments, the diimide is1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. In some embodiments, themethod includes reacting the precursor represented by formula (IV) withthe precursor represented by formula (V) and a benzotriazole derivative,such as a benzotriazole derivative selected from the group consisting of1-hydroxybenzotriazole, 6-chloro-1-hydroxybenzotriazole, and1-hydroxy-7-azabenzotriazole. In some embodiments, the benzotriazolederivative is 1-hydroxybenzotriazole.

In some embodiments, the method includes reacting the precursorrepresented by formula (IV) with the precursor represented by formula(V) and a base, such as N,N-dimethylaminopyridine.

In some embodiments, the method includes synthesizing the precursorrepresented by formula (IV) by reacting a precursor represented byformula (VI)

with a precursor represented by formula (VII).

In some embodiments, the method includes reacting the precursorrepresented by formula (VI) with the precursor represented by formula(VII) and one or more bases. In some embodiments, the one or more basesare selected from the group consisting of diisopropylethylamine,triethylamine, and N,N-dimethylaminopyridine.

In some embodiments, the method includes reacting the precursorrepresented by formula (VI) with the precursor represented by formula(VII), diisopropylethylamine, and N,N-dimethylaminopyridine.

In an additional aspect, the invention provides a method of making acompound represented by formula (III),

wherein the method includes mixing a compound represented by formula (I)

with hydrochloric acid.

In some embodiments, the hydrochloric acid is aqueous hydrochloric acid.The aqueous hydrochloric acid may be prepared, for instance, by dilutingthe hydrochloric acid in water, such as distilled or deionized water. Insome embodiments, the method includes making the compound represented byformula (III) in a crystalline state.

In some embodiments, the method includes dissolving the compoundrepresented by formula (I) in ethanol. In some embodiments, the methodincludes mixing the hydrochloric acid with ethanol. In some embodiments,the method includes mixing the hydrochloric acid with ethyl acetate. Insome embodiments, the method includes adding the compound represented byformula (I) to the hydrochloric acid over a period of from about 20 toabout 30 minutes to form a mixture. In some embodiments, the methodincludes maintaining the temperature of the mixture at from about 15° C.to about 25° C. during the adding. In some embodiments, the methodincludes reducing the temperature of the mixture to about 5° C.following the adding. In some embodiments, the method includes stirringthe mixture for from about 50 to about 70 minutes at from about 0° C. toabout 5° C. following the reducing.

In some embodiments, the method includes mixing the compound representedby formula (I) and the hydrochloric acid in equimolar amounts.

In another aspect, the invention encompasses a compound produced by anyof the above-described methods.

In an additional aspect, the invention provides a method of treating orpreventing preterm labor in a subject by administering to the subject atherapeutically effective amount of the compound or pharmaceuticalcomposition of any of the above-described aspects of the invention.

In another aspect, the invention provides a method of preventing laborprior to cesarean delivery in a subject by administering to the subjecta therapeutically effective amount of the compound or pharmaceuticalcomposition of any of the above-described aspects of the invention.

In another aspect, the invention provides a method of treating orpreventing dysmenorrhea in a subject by administering to the subject atherapeutically effective amount of the compound or pharmaceuticalcomposition of any of the above-described aspects of the invention.

In another aspect, the invention provides a method of treating orpreventing endometriosis in a subject by administering to the subject atherapeutically effective amount of the compound or pharmaceuticalcomposition of any of the above-described aspects of the invention.

In some embodiments, the subject is characterized by a gestational ageof from about 24 to about 34 weeks. In some embodiments, the subjectexhibits a reduction in the amplitude of uterine contractions followingthe administering, such as a reduction of by from about 40% to about 50%(e.g., about 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%)relative to a measurement of the amplitude of uterine contractions inthe subject recorded prior to the administering. In some embodiments,the compound exhibits a half life of from about 1 to about 4 hours inthe subject (e.g., about 1 hour, 1.1 hours, 1.2 hours, 1.3 hours, 1.4hours, 1.5 hours, 1.6 hours, 1.7 hours, 1.8 hours, 1.9 hours, 2.0 hours,2.1 hours, 2.2 hours, 2.3 hours, 2.4 hours, 2.5 hours, 2.6 hours, 2.7hours, 2.8 hours, 2.9 hours, 3.0 hours, 3.1 hours, 3.2 hours, 3.3 hours,3.4 hours, 3.5 hours, 3.6 hours, 3.7 hours, 3.8 hours, 3.9 hours, or 4.0hours). In some embodiments, the compound reaches a maximum plasmaconcentration in the subject within from about 0.25 to about 2 hours ofthe administering (e.g., about 0.25 hours, 0.3 hours, 0.4 hours, 0.5hours, 0.6 hours, 0.7 hours, 0.8 hours, 0.9 hours, 1.0 hours, 1.1 hours,1.2 hours, 1.3 hours, 1.4 hours, 1.5 hours, 1.6 hours, 1.7 hours, 1.8hours, 1.9 hours, or 2.0 hours). In some embodiments, the subject is amammal, such as a human.

In some embodiments, the method includes orally administering thecompound or pharmaceutical composition to the subject. In someembodiments, the method includes intravenously administering thecompound or pharmaceutical composition to the subject. In someembodiments, the compound is administered to the subject in combinationwith an oxytocin receptor antagonist. In some embodiments, the methodincludes orally administering the oxytocin receptor antagonist to thesubject. In some embodiments, the method includes intravenouslyadministering the oxytocin receptor antagonist to the subject. Thecompound may be administered to the subject at the same time as theoxytocin receptor antagonist is administered. Alternatively, thecompound may be administered to the subject before administration of theoxytocin receptor antagonist to the subject. In some embodiments, thecompound is administered to the subject after administration of theoxytocin receptor antagonist to the subject. In some embodiments, thecompound is admixed with the oxytocin receptor antagonist, and theseagents are administered to the subject concurrently. In someembodiments, the oxytocin receptor antagonist is atosiban, retosiban,barusiban, epelsiban, or nolasiban, or a derivative thereof.

In some embodiments, the invention provides a kit containing thecompound or pharmaceutical composition of any of the above-describedaspects of the invention, as well as a package insert. In someembodiments, the package insert instructs a user of the kit toadminister the compound or pharmaceutical composition to a subjectpresenting with preterm labor. In some embodiments, the subject ischaracterized by a gestational age of from about 24 to about 34 weeks.In some embodiments, the package insert instructs a user of the kit tomix the compound or pharmaceutical composition with an aqueous solution.In some embodiments, the package insert instructs a user of the kit toorally administer the compound to the subject.

DEFINITIONS

As used herein, the term “about” refers to a value that is within 10%above or below the value being described.

As used herein, the term “affinity” refers to the strength of a bindinginteraction between two molecules, such as a ligand and a receptor. Theterm “K_(i)”, as used herein, is intended to refer to the inhibitionconstant of an antagonist for a particular molecule of interest, and isexpressed as a molar concentration (M). K_(i) values forantagonist-target interactions can be determined, e.g., using methodsestablished in the art. Methods that can be used to determine the K_(i)of an antagonist for a molecular target include competitive bindingexperiments, such as competitive radioligand binding assays, e.g., asdescribed in U.S. Pat. No. 8,415,480. The term “K_(d)”, as used herein,is intended to refer to the dissociation constant, which can beobtained, e.g., from the ratio of the rate constant for the dissociationof the two molecules (k_(d)) to the rate constant for the association ofthe two molecules (k_(a)) and is expressed as a molar concentration (M).K_(d) values for receptor-ligand interactions can be determined, e.g.,using methods established in the art. Methods that can be used todetermine the K_(d) of a receptor-ligand interaction include surfaceplasmon resonance, e.g., through the use of a biosensor system such as aBIACORE® system.

As used herein, the term “crystalline” or “crystalline form” meanshaving a physical state that is a regular three-dimensional array ofatoms, ions, molecules or molecular assemblies. Crystalline forms havelattice arrays of building blocks called asymmetric units that arearranged according to well-defined symmetries into unit cells that arerepeated in three-dimensions. In contrast, the term “amorphous” or“amorphous form” refers to an unorganized (no orderly) structure. Thephysical state of a therapeutic compound may be determined by exemplarytechniques such as x-ray diffraction, polarized light microscopy and/ordifferential scanning calorimetry.

As used herein, the term “endogenous” describes a molecule (e.g., apolypeptide, nucleic acid, or cofactor) that is found naturally in aparticular organism (e.g., a human) or in a particular location withinan organism (e.g., an organ, a tissue, or a cell, such as a human cell).

As used herein, the term “exogenous” describes a molecule (e.g., apolypeptide, nucleic acid, or cofactor) that is not found naturally in aparticular organism (e.g., a human) or in a particular location withinan organism (e.g., an organ, a tissue, or a cell, such as a human cell).Exogenous materials include those that are provided from an externalsource to an organism or to cultured matter extracted therefrom.

As used herein, the term “gestational age” describes how far along aparticular pregnancy is, and is measured from the first day of apregnant female subject's last menstrual cycle to the current date. Asused herein, the term “labor” (which may also be termed birth) relatesto the expulsion of the fetus and placenta from the uterus of a pregnantfemale subject. For a normal pregnancy, labor may occur at a gestationalage of about 40 weeks. “Preterm labor” as used herein refers to acondition in which labor commences more than three weeks before the fullgestation period, which is typically about 40 weeks. That is, pretermlabor occurs at any stage prior to, e.g., 38 weeks of gestation. Pretermlabor typically leads to the occurrence of labor, or physiologicalchanges associated with labor in a pregnant female subject, if nottreated. Preterm labor may or may not be associated with vaginalbleeding or rupture of uterine membranes. Preterm labor may also bereferred to as premature labor. The avoidance of preterm labor in asubject will prolong the term of pregnancy and may therefore avoidpreterm delivery, thus reducing the risk of neonatal mortality andmorbidity.

As used herein, the term “IC₅₀” refers to the concentration of asubstance (antagonist) that reduces the efficacy of a reference agonistor the constitutive activity of a biological target by 50%, e.g., asmeasured in a competitive ligand binding assay. Exemplary competitiveligand binding assays include competitive radioligand binding assays,competitive enzyme-linked immunosorbent assays (ELISA), and fluorescenceanisotropy-based assays, among others known in the art.

As used herein, the term “oral bioavailability” refers to the fractionof a compound administered to a subject, such as a mammal (e.g., ahuman) that reaches systemic circulation in the subject, and that is notsequestered in a non-target organ or excreted without absorption via thegastrointestinal tract. The term refers to a blood plasma concentrationthat is integrated over time and is typically expressed as a percentageof the orally administered dose.

As used herein, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions and/or dosage forms, which aresuitable for contact with the tissues of a subject, such as a mammal(e.g., a human) without excessive toxicity, irritation, allergicresponse and other problem complications commensurate with a reasonablebenefit/risk ratio.

As used herein, the term “pharmaceutical composition” means a mixturecontaining a therapeutic compound to be administered to a subject, suchas a mammal, e.g., a human, in order to prevent, treat or control aparticular disease or condition affecting the mammal, such as pretermlabor or dysmenorrhea.

As used herein, the term “protecting group” refers to a chemical moietywhich, when bound to a functional group, renders the functional groupinert to one or more chemical reactions. Such reactions may modify oneor more substituents of the compound and, in the absence of a protectinggroup, might result in undesired chemical modification (e.g.,electrophilic addition, solovolysis, oxidation, reduction, or functionalgroup interconversion) of a moiety of interest (e.g., an amino,hydroxyl, carboxyl, or carboxamide moiety). Protecting groups may, atthe appropriate time, be chemically reacted so as to regenerate theoriginal functionality. The identity of the protecting group can beselected so as to be compatible with the remainder of the molecule,e.g., such that the protecting group is not removed during other stepsof the synthesis or modification of the molecule, and optionally, suchthat the reaction conditions used to effect the removal of theprotecting group do not result in the removal of different protectinggroups located at other substituents on the molecule. Exemplaryprotecting groups include those that can be covalently bound to, e.g.,an amino substituent, such as the amino group of an α-amino ester. Thesubsequent removal of a protecting group, referred to herein as the“deprotection” of a chemical moiety, can be achieved using reagents andconditions known in the art. Examples of protecting groups include,without limitation, benzyl, acetyl, oxyacetyl, carboxybenzyl,9-fluorenyloxycarbonyl, 2-chloro-1-indanylmethoxy-carbonyl,benz[f]indene-3-methoxycarbonyl,2-(tert-butylsulfonyl)-2-propenyloxycarbonyl, benzothiophenesulfone-2-methylcarbonyl, tert-butoxycarbonyl, tert-amyloxycarbonyl,β-trimethylsilylethyloxycarbonyl, adamantyloxycarbonyl,1-methylcyclobutyloxycarbonyl, 2-(p-biphenylyl)propyl-2-oxycarbonyl,2-(p-phenylazophenyl)propyl-2-oxycarbonyl,2-2-dimethyl-3,5-dimethyloxybenzyloxycarbonyl,2-phenylpropyl-2-oxycarbonyl, benzyloxycarbonyl,p-toluenesulfonylaminocarbonyl, o-nitrophenylsulfenyl, dithiasuccinoyl,phthaloyl, piperidinooxycarbonyl, formyl, trifluoroacetyl,2,4,6-trimethoxybenzyl, 2,3,6-trimethyl-4 methoxybenzenesulfonyl,tert-butoxymethyl, pentamethylchromanesulfonyl, adamantly,β-trimethylsilylethyl, β-trimethylilylethyloxycarbonyl, tert-butyl,tert-butylbenzyl, cyclopentyl, triphenylmethyl, benzyloxycarbonyl,formyl, and trifluoroacetyl, among others. Protecting groups may besuitable for a particular chemical substituent. For instance, examplesof hydroxyl protecting groups include, without limitation, benzyl,p-methoxybenzyl, p-nitrobenzyl, allyl, trityl, dialkylsilylethers, suchas dimethylsilyl ether, and trialkylsilyl ethers such as trimethylsilylether, triethylsilyl ether, and t-butyldimethylsilyl ether; esters suchas benzoyl, acetyl, phenylacetyl, formyl, mono-, di-, and trihaloacetylsuch as chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl;and carbonates such as methyl, ethyl, 2,2,2-trichloroethyl, allyl,benzyl, and p-nitrophenyl. Additional examples of protecting groups maybe found, e.g., in Greene and Wuts, Protective Groups in OrganicSynthesis, 2d Ed., 1991, John Wiley & Sons, as well as in McOmie,Protective Groups in Organic Chemistry, 1975, Plenum Press, thedisclosures of each of which are incorporated herein by reference. Otherexamples of protecting groups are described, e.g., in U.S. Pat. Nos.3,835,175; 4,508,657; 3,839,396; 4,581,167; 4,460,501; and 4,108,846,the disclosures of each of which are incorporated herein by reference.

As used herein, the term “sample” refers to a specimen (e.g., blood,blood component (e.g., serum or plasma), urine, saliva, amniotic fluid,cerebrospinal fluid, tissue (e.g., placental or dermal), pancreaticfluid, chorionic villus sample, and cells) isolated from a subject.

As used herein, the phrases “specifically binds” and “binds” refer to abinding reaction which is determinative of the presence of a particularprotein in a heterogeneous population of proteins and other biologicalmolecules that is recognized, e.g., by a ligand with particularity. Aligand (e.g., a protein, proteoglycan, or glycosaminoglycan) thatspecifically binds to a protein will bind to the protein, e.g., with aK_(D) of less than 100 nM. For example, a ligand that specifically bindsto a protein may bind to the protein with a K_(D) of up to 100 nM (e.g.,between 1 pM and 100 nM). A ligand that does not exhibit specificbinding to a protein or a domain thereof will exhibit a K_(D) of greaterthan 100 nM (e.g., greater than 200 nM, 300 nM, 400 nM, 500 nM, 600 nm,700 nM, 800 nM, 900 nM, 1 μM, 100 μM, 500 μM, or 1 mM) for thatparticular protein or domain thereof. A variety of assay formats may beused to determine the affinity of a ligand for a specific protein. Forexample, solid-phase ELISA assays are routinely used to identify ligandsthat specifically bind a target protein. See, e.g., Harlow & Lane,Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York(1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, ColdSpring Harbor Press, New York (1999), for a description of assay formatsand conditions that can be used to determine specific protein binding.

As used herein, the terms “subject” and “patient” are interchangeableand refer to an organism that receives treatment for a particulardisease or condition as described herein (such as preterm labor ordysmenorrhea) or that is diagnosed as having a disease or conditionaccording to the methods described herein. Examples of subjects andpatients include mammals, such as humans, receiving treatment fordiseases or conditions, for example, preterm labor at an earlygestational age (e.g., 24-34 weeks).

A compound, salt form, crystal polymorph, therapeutic agent, or othercomposition described herein may be referred to as being characterizedby graphical data “substantially as depicted in” a figure. Such data mayinclude, without limitation, powder X-ray diffractograms, NMR spectra,differential scanning calorimetry curves, and thermogravimetric analysiscurves, among others. As is known in the art, such graphical data mayprovide additional technical information to further define the compound,salt form, crystal polymorph, therapeutic agent, or other composition.As is understood by one of skill in the art, such graphicalrepresentations of data may be subject to small variations, e.g., inpeak relative intensities and peak positions due to factors such asvariations in instrument response and variations in sample concentrationand purity. Nonetheless, one of skill in the art will readily be capableof comparing the graphical data in the figures herein with graphicaldata generated for a compound, salt form, crystal polymorph, therapeuticagent, or other composition and confirm whether the two sets ofgraphical data are characterizing the same material or two differentmaterials. For instance, a crystal form of(3S)-3-({[(2S)-3-(biphenyl-4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3-(4-fluorophenyl)propylL-valinate hydrochloride referred to herein as being characterized bygraphical data “substantially as depicted in” a figure will thus beunderstood to include any crystal form of(3S)-3-({[(2S)-3-(biphenyl-4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3-(4-fluorophenyl)propylL-valinate hydrochloride characterized by the graphical data, optionallyhaving one or more of small variations, e.g., one or more variationsdescribed above or known to one of skill in the art.

As used herein, the terms “treat” or “treatment” refer to therapeutictreatment, in which the object is to prevent or slow down (lessen) anundesired physiological change or disorder, such as the progression ofpreterm labor at an early gestational age (e.g., 24-34 weeks).Beneficial or desired clinical results include, but are not limited to,alleviation of symptoms, such as vaginal bleeding or membrane rupture,and the delay or slowing of labor. Those in need of treatment include,e.g., pregnant female subjects already experiencing preterm labor, aswell as those prone to developing this condition.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph demonstrating the effect of compound II and compoundIII on spontaneous uterine contractility in late-term pregnant ratsfollowing intravenous administration.

FIG. 2 is a graph showing the dose-dependent and reversible effect ofcompound I on spontaneous uterine contraction in late-term pregnantrats.

FIG. 3 is a graph demonstrating the effect of compound II and compoundIII on spontaneous uterine contractility in late-term pregnant ratsfollowing oral administration.

FIG. 4 is a table summarizing various methods used to generate the freebase of compound I, as well as observations regarding the physicalcharacteristics and NMR spectra of compound I as generated by eachmethod.

FIG. 5 is a table summarizing various methods used to generate salts ofcompound I, as well as observations regarding the physicalcharacteristics and NMR spectra of these salts as generated by eachmethod.

FIG. 6 is a table summarizing physical characteristics as well as X-raypowder diffraction (XRPD) spectra of various salts of compound I.

FIG. 7 is a table summarizing methods used to generate crystal forms ofvarious compound I salts, as well as observations regarding the physicalproperties and XRPD spectra of each crystal form.

FIG. 8 is a table summarizing the solubility of various compound I saltsin aqueous solution.

FIG. 9 is a table summarizing the stability of crystal forms of variouscompound I salts at the indicated relative humidity (RH).

FIG. 10 is a table summarizing various characteristics of compound IIIas determined by X-ray powder diffraction (XRPD), differential scanningcalorimetry (DSC), thermogravimetric (TG) analysis, moisturesorption/desorption (MB), and ¹H nuclear magnetic resonance (NMR).

FIG. 11 is a table summarizing various characteristics of thehydrosulfate salt of compound I as determined by X-ray powderdiffraction (XRPD), differential scanning calorimetry (DSC),thermogravimetric (TG) analysis, and ¹H nuclear magnetic resonance(NMR).

FIG. 12 shows an XRPD spectrum of the mesylate salt of compound I.

FIG. 13 shows a ¹H NMR spectrum of the mesylate salt of compound I.

FIG. 14 shows an XRPD spectrum of the free base of compound I.

FIG. 15 shows a ¹H NMR spectrum of the free base of compound I.

FIG. 16 shows a Raman infrared spectrum of the free base of compound I.

FIG. 17 shows a ¹H NMR spectrum of the mesylate salt of compound I. Themesylate salt was prepared by addition of methanesulfonic acid to asolution of the free base of compound I in diethyl ether.

FIG. 18 shows a series of ¹H NMR spectra of the free base of compound Irecorded during homonuclear decoupling experiments.

FIG. 19 shows a series of XRPD spectra of the chloride salt of compoundI as produced from an acetone slurry (top), from evaporation of amethylene chloride:ethyl ether mixture (second from top), and from slowevaporation of a 1:1 acetone:toluene mixture (second from bottom andbottom).

FIG. 20 shows an overlay of a differential scanning calorimetry curve(ranging from about −0.5 to about 1.3 W/g) and a thermogravimetricanalysis curve (ranging from about 0% to about 100% by weight) recordedfor the chloride salt of compound I as produced from an acetone slurry.

FIG. 21 shows a ¹H NMR spectrum of the chloride salt of compound I asproduced from a 1:1 acetone:toluene mixture.

FIG. 22 shows a series of XRPD spectra of the chloride salt of compoundI as produced from an acetone slurry (top) and after being vacuum-driedat about 50° C. for 1 day (bottom).

FIG. 23 shows an overlay of a differential scanning calorimetry curve(ranging from about −1.0 to about 0.2 W/g) and a thermogravimetricanalysis curve (ranging from about 30% to about 100% by weight) recordedfor the chloride salt of compound I after being vacuum-dried at about50° C. for 1 day.

FIG. 24 shows an overlay of thermogravimetric analysis curves of thechloride salt of compound I as produced from an acetone slurry (top) andafter being vacuum-dried at about 50° C. for 1 day (bottom).

FIG. 25 shows an overlay of differential scanning calorimetry curvesrecorded for the chloride salt of compound I as produced from an acetoneslurry (top) and after being vacuum-dried at about 50° C. for 1 day(bottom).

FIG. 26 shows a moisture sorption/desorption curve recorded for thechloride salt of compound I. Values on the y-axis show the percentchange in the weight of the chloride salt as a function of the relativehumidity (RH) in the atmosphere surrounding the salt.

FIG. 27 is a table reporting the data obtained from moisturesorption/desorption experiments performed with the chloride salt ofcompound I.

FIG. 28 shows a moisture sorption/desorption curve recorded for thechloride salt of compound I. Values on the y-axis show the percentchange in the weight of the chloride salt as a function of the time overwhich the relative humidity in the atmosphere surrounding the salt wasaltered.

FIG. 29 shows an overlay of XRPD spectra of the chloride salt ofcompound I following (top) and prior to performing (bottom) moisturesorption/desorption experiments.

FIG. 30 shows an overlay of an XRPD spectrum of the fumarate salt ofcompound I produced by slow evaporation of a 1:1 methanol:toluenemixture (top) and an XRPD of fumaric acid (bottom).

FIG. 31 shows an overlay of an XRPD spectrum of the dihydrophosphatesalt of compound I (top) and an XRPD of the hydrosulfate salt ofcompound I (bottom).

FIG. 32 shows an overlay of a differential scanning calorimetry curve(ranging from about −1.9 to about 0 W/g) and a thermogravimetricanalysis curve (ranging from about 25% to about 95% by weight) recordedfor the hydrosulfate salt of compound I.

FIG. 33 shows a ¹H NMR spectrum of the hydrosulfate salt of compound I.

FIG. 34 shows a ¹H NMR spectrum of the sulfate salt of compound I.

FIG. 35 shows an XRPD spectrum of the mesylate salt of compound I.

FIG. 36 shows an XRPD spectrum of the citrate salt of compound I.

FIG. 37 shows an XRPD spectrum of the edisylate salt of compound I.

FIG. 38 shows an XRPD spectrum of the hydrosulfate salt of compound I.

FIG. 39 shows an XRPD spectrum of the citrate salt of compound I asproduced by slow evaporation of a 1:2 methanol:toluene mixture.

FIG. 40 shows an XRPD spectrum of the hydrosulfate salt of compound I asproduced by slow evaporation of a 6:1 ethyl acetate:heptane mixture.

FIG. 41 shows an XRPD spectrum of the hydrosulfate salt of compound I asproduced by slow evaporation of an ethyl acetate mixture.

FIG. 42 shows an XRPD spectrum of the dihydrophosphate salt of compoundI as produced by slow evaporation of a 1:2 methanol:acetonitrilemixture.

FIG. 43 shows an XRPD spectrum of the dihydrophosphate salt of compoundI as produced by slow evaporation of a 1:1 methyl ethyl ketone:n-butylacetate mixture.

FIG. 44 shows an XRPD spectrum recorded from a duplicate XRPD experimentof the dihydrophosphate salt of compound I as produced by slowevaporation of a 1:1 methyl ethyl ketone:n-butyl acetate mixture.

FIG. 45 shows an XRPD spectrum of the chloride salt of compound I asproduced by slow evaporation of a 1:1 acetone:toluene mixture.

FIG. 46 shows an XRPD spectrum recorded from a duplicate XRPD experimentof the chloride salt of compound I as produced by slow evaporation of a1:1 acetone:toluene mixture.

FIG. 47 shows an XRPD spectrum of the chloride salt of compound I asproduced by slow evaporation of a diethyl ether:methylene chloridemixture.

FIG. 48 shows an XRPD spectrum of the chloride salt of compound I asproduced from an acetone slurry.

FIG. 49 shows an XRPD spectrum of the chloride salt of compound I afterbeing vacuum dried.

FIG. 50 shows an XRPD spectrum of the fumarate salt of compound I asproduced by slow evaporation of a 1:1 methanol:toluene mixture.

FIG. 51 shows an XRPD spectrum of the fumarate salt of compound I asproduced by slow evaporation of a 1:1 methanol:ethyl acetate mixture.

FIG. 52 shows an XRPD spectrum of the fumarate salt of compound I asproduced by vacuum drying a 1:1 methanol:toluene mixture.

FIG. 53 shows an XRPD spectrum of the edisylate salt of compound I asproduced by slow evaporation of a 1:1:1 methanol:methyl ethylketone:toluene mixture.

FIG. 54 shows an overlay of XRPD spectra of the chloride salt ofcompound I prior to (bottom) and following (top) storage at 40° C. and75% relative humidity.

FIG. 55 is a table summarizing the stability of the mesylate salt ofcompound I and compound II in the buffer used in Caco-2 penetrationexperiments: Hank's Balanced Salt Solution (HBSS) buffer, 2% finalconcentration of DMSO.

FIG. 56a is a table reporting data obtained from analysis of the abilityof the mesylate salt of compound I to pass from the apical to thebasolateral compartment of a transwell coated with a Caco-2 cellmonolayer. Cultured Caco-2 cells were incubated with the indicatedconcentration of the mesylate salt of compound I in the apicalcompartment of the transwell, and aliquots from the basolateralcompartment were sampled at the indicated sampling times in order todetermine the presence of compound I or compound II. The data reportsthe concentration of compound II in the basolateral compartment as apercentage of the indicated initial concentration of the mesylate saltof compound I. FIG. 56b is a table reporting data obtained from analysisof the ability of the mesylate salt of compound I to pass from thebasolateral to the apical compartment of a transwell coated with aCaco-2 cell monolayer. Cultured Caco-2 cells were incubated with theindicated concentration of the mesylate salt of compound I in thebasolateral compartment of the transwell, and aliquots from the apicalcompartment were sampled at the indicated sampling times in order todetermine the presence of compound I or compound II. The data reportsthe concentration of compound II in the basolateral compartment as apercentage of the indicated initial concentration of the mesylate saltof compound I. FIG. 56c is a graph showing the relative concentration ofcompound II in the basolateral compartment as a percentage of theinitial concentration of the mesylate salt of compound I in the apicalcompartment. FIG. 56d is a graph showing the relative concentration ofcompound II in the apical compartment as a percentage of the initialconcentration of the mesylate salt of compound I in the basolateralcompartment. Compound I was not detected in the basolateral compartmentfollowing 60 or 120 minutes of incubation in the apical compartment.Additionally, compound I was not detected in the apical compartmentfollowing 60 or 120 minute of incubation in the basolateral compartment.Rather, compound II was detected in each case. FIG. 56e is a tableshowing the recovery of compound I in the apical compartment following120 minutes of incubation. The initial compound was primarily recoveredin the form of the de-esterified variant, compound II.

FIG. 57a is a table reporting data obtained from analysis of the abilityof compound II to pass from the apical to the basolateral compartment ofa transwell coated with a Caco-2 cell monolayer. Cultured Caco-2 cellswere incubated with the indicated concentration of compound II in theapical compartment of the transwell, and aliquots from the basolateralcompartment were sampled at the indicated sampling times in order todetermine the presence of compound II. The data reports theconcentration of compound II in the basolateral compartment as apercentage of the indicated initial concentration of compound II. FIG.57b is a table reporting data obtained from analysis of the ability ofcompound II to pass from the basolateral to the apical compartment of atranswell coated with a Caco-2 cell monolayer. Cultured Caco-2 cellswere incubated with the indicated concentration of compound II in thebasolateral compartment of the transwell, and aliquots from the apicalcompartment were sampled at the indicated sampling times in order todetermine the presence of compound II. The data reports theconcentration of compound II in the basolateral compartment as apercentage of the indicated initial concentration of compound II. FIG.57c is a table showing the recovery of compound II in the apicalcompartment following 60 and 120 minutes of incubation in thebasolateral compartment, as well as the permeability rate of compound IIthrough the Caco-2 cell monolayer. FIG. 57d is a graph showing therelative concentration of compound II in the basolateral compartment asa percentage of the initial concentration of compound II in the apicalcompartment. FIG. 57e is a graph showing the relative concentration ofcompound II in the apical compartment as a percentage of the initialconcentration of compound II I in the basolateral compartment.

FIG. 58a is a table reporting data obtained from analysis of the abilityof the mesylate salt of compound I to pass from the apical to thebasolateral compartment of a transwell coated with a Caco-2 cellmonolayer. Cultured Caco-2 cells were incubated with the indicatedconcentration of the mesylate salt of compound I in the apicalcompartment of the transwell, and aliquots from the basolateralcompartment were sampled at the indicated sampling times in order todetermine the presence of compound I or compound II. The data reportsthe concentration of compound II in the basolateral compartment as apercentage of the indicated initial concentration of the mesylate saltof compound I. Compound I was not detected in the basolateralcompartment following 60 or 120 minutes of incubation in the apicalcompartment. FIG. 58b is a graph showing the relative concentration ofcompound II in the basolateral compartment as a percentage of theinitial concentration of the mesylate salt of compound I in the apicalcompartment. FIG. 58c is a table showing the recovery of compound I inthe apical compartment following 120 minutes of incubation. The initialcompound was primarily recovered in the form of the de-esterifiedcompound variant, compound II.

FIG. 59 is a table summarizing the chromatography and mass spectrometryparameters used for the analysis of concentrations of compound I andcompound II in Caco-2 cell penetration experiments described herein.

DETAILED DESCRIPTION

The invention provides α-amino esters of a thiazolidine carboxamide,such as(3S)-3-({[(2S)-3-(biphenyl-4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3-(4-fluorophenyl)propylL-valinate, as well as salt forms and crystal polymorphs thereof. Thesecompounds are capable of inhibiting the activity of proteins of theprostaglandin F receptor (FP-R) family, such as prostaglandin F2α(PGF2α) receptor. The compounds, salts, and crystal polymorphs describedherein can be used to inhibit the activity of the prostaglandin Freceptor in vitro and in vivo, and represent effective therapeuticcompositions for the treatment of preterm labor. The compounds, salts,and crystal polymorphs described herein can be administered to a subject(e.g., a mammalian subject, such as a human) that is undergoing or is atrisk of undergoing labor at an early gestational age, e.g., prior to 38weeks (e.g., from about 20 to about 37 weeks, such as a gestational ageof about 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33weeks, 34 weeks, 35 weeks, 36 weeks, or 37 weeks, preferably from about24 to about 34 weeks, such as a gestational age of about 24 weeks, 25weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32weeks, 33 weeks, or 34 weeks. The invention additionally providesmethods of synthesizing(3S)-3-({[(2S)-3-(biphenyl-4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3-(4-fluorophenyl)propylL-valinate, as well as processes for preparing salt forms and crystalpolymorphs thereof. The invention further encompasses methods oftreating preterm labor in a subject by administering an alpha-aminoester of the invention to a subject in need of treatment, such as asubject experiencing preterm labor or a subject at risk of undergoingpreterm labor.

(3S)-3-({[(2S)-3-(biphenyl-4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3-(4-fluorophenyl)propylL-valinate (Compound I)

The invention is based on the discovery that compound I((3S)-3-({[(2S)-3-(biphenyl-4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3-(4-fluorophenyl)propylL-valinate, represented by formula I, below) and salts thereof areconverted in vivo to3-([1,1′-biphenyl]-4-ylsulfonyl)-N-[1-(4-fluorophenyl)-3-hydroxypropyl]-1,3-thiazolidine-2-carboxamide(represented by formula II, below). Compound II, previously described inU.S. Pat. No. 8,415,480, is an antagonist of the prostaglandin Freceptor, as this compound exhibits an inhibition constant (Ki) of 6 nMfor human FP-R as determined by competitive radioligand binding assays(experimental details of competitive radioligand binding assays usefulfor the determination of Ki values are described, e.g., in U.S. Pat. No.8,415,480, Example 51). Following administration to a subject, compoundI has been found to be de-esterified in vivo so as to form compound IIdue to the activity of endogenous esterases, such as those present inthe gastrointestinal tract.

It has been discovered that compound I is an inhibitor of theprostaglandin F receptor, as compound I inhibits human FP-R with a Ki of1 nM. Compound I exhibits improvements in several physicochemicalcharacteristics relative to compound II, including solubility in wateras well as in media that simulate the small intestinal contents in thefed (FeSSIF) and fasted (FaSSIF) states. These data are summarized inTable 1, below.

TABLE 1 Comparison of physicochemical properties of compound I andcompound II Parameter Compound I Compound II Solubility in water (μg/mL)380 0.4 Solubility in FaSSIF (μg/mL) pH 6.5 70 0.4 Solubility in FeSSIF(μg/mL) pH 5.0 90 10 Human FP-R Ki (nM) 1 6

(3S)-3-({[(2S)-3-(biphenyl-4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3-(4-fluorophenyl)propylL-valinate hydrochloride (Compound III)

It has been discovered that the chloride salt of compound I((3S)-3-({[(2S)-3-(biphenyl-4-ylsulfonyl)-1,3-thiazolidin-2-yl]carbonyl}-amino)-3-(4-fluorophenyl)propylL-valinate hydrochloride, designated as formula III below) is readilycrystallized using a several distinct experimental procedures, asdescribed in the Examples below. Compound III assumes a single,reproducible crystal form upon crystallization from a variety of mediaand under different ambient conditions. Moreover, this crystal form ofcompound III exhibits extended stability under ambient conditions and inthe presence of elevated relative humidity. As is described in furtherdetail in the Examples presented below, compound III exhibits a lowhygroscopicity and thus does not demonstrate a propensity to absorbmoisture from the local atmosphere. Compound III therefore exhibits aresistance to chemical changes, such as hydrolysis, as well as aresistance to the incorporation of impurities. For instance, impuritiesassociated with atmospheric water are not readily integrated into thecrystalline form of compound III. Compound III can be administered to asubject, such as a pregnant female human subject, in order to delay theonset of labor in a subject, e.g., by one or more days or weeks, such asfrom about 1 day to about 16 weeks (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30 days, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, or 16 weeks). Compound III also be administered to a subject, suchas a pregnant female human subject, in order to alleviate one or moresymptoms associated with labor, such as vaginal bleeding and rupture ofuterine membranes. Compound III may be administered alone or incombination with one or more additional agents, such as an oxytocinreceptor antagonist described herein (e.g., nolasiban, which is (3Z,5S)-5-(hydroxymethyl)-1-[(2′-methyl-1,1′-biphenyl-4-yl)carbonyl]pyrrolidin-3-oneO-methyl oxime, or a derivative thereof). Additionally, compound III maybe formulated into a pharmaceutical composition, such as apharmaceutical composition formulated as described below.

Methods of Treatment

Compound I, as well as salts thereof, represent robust inhibitors of theprostaglandin F receptor and can be used to antagonize the interactionbetween prostaglandin F family members, such as prostaglandin F2α, withthe corresponding prostaglandin F receptor in vivo in order to attenuateuterine contractions. Compound I and salts thereof can be administeredto a subject, such as a pregnant human female subject, in order to treator prevent preterm labor.

Endogenous prostaglandin F2α is synthesized in and released by uterineepithelial cells in response to the signal transduction cascadesinitiated by oxytocin. Upon binding of PGF2α to PGF2α-R on theextracellular surface of a uterine myocyte, phospholipase C cleavesphosphatidylinsolitol-4,5-bisphosphate (PIP₂) to yield diacylglycerol(DAG) and inositol-1,4,5-trisphosphate (IP₃). IP₃ in turn potentiatesthe release of intracellular calcium (Ca²⁺) sarcoplasmic reticula. Thesudden increase in calcium stores ultimately leads to uterine musclecontractions and a necrosis of endothelial cells of the corpus luteum, aprogesterone-secreting structure that supports a developing fetus. Theaberrant initiation of uterine contractions and degradation of thecorpus luteum caused by dysregulation of PGF2α secretion can lead topreterm labor. Compound I and salts thereof, such as compound III, mayattenuate the phospholipase C-mediated formation of IP₃, and thesubsequent mobilization of intracellular calcium stores, by inhibitingthe association of PGF2α with the PGF2αR. Compound I or a salt thereof,such as compound III, can thus be administered to subjects, such aspregnant female human subjects, in order to delay the onset of labor ina subject, e.g., by one or more days or weeks, such as from about 1 dayto about 16 weeks (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days,or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16weeks). For instance, compound I or a salt thereof, such as compoundIII, can be administered to a subject in order to prevent labor prior tocesarean delivery. Additionally, compound I or a salt thereof, such ascompound III, can be administered to a subject for the prophylaxisand/or treatment of dysmenorrhea. Compound I or a salt thereof, such ascompound III, can also be administered to a subject, such as a pregnantfemale human subject, in order to alleviate one or more symptomsassociated with labor, such as vaginal bleeding and rupture of uterinemembranes.

Additionally, compounds of the invention can be used to treatendometriosis in a patient (e.g., a human patient). Prostaglandin F2αreceptor overexpression has been correlated with aberrant endometrialgrowth. As antagonists of prostaglandin F2α receptor activity, thecompounds of the invention (e.g., compound (I) or a salt thereof, suchas compound (III)) can be administered to a patient suffering fromendometriosis in order to treat this indication. The compounds of theinvention can also be administered to a patient in order to alleviateone or more symptoms of endometriosis, such pain symptoms includingdysmenorrhea, dyspareunia, chronic pelvic pain, dysuria, and dyscheziaduring and/or apart from menstruation. Successful treatment ofendometriosis by administration of a compound of the invention to apatient can be indicated by, e.g., a reduction in the growth ofendometrial tissue, and/or a reduction in pain symptoms during and/orapart from menstruation.

Combination Therapy

Though the processes involved in the onset of labor are not yet fullydefined, there is increasing evidence supporting the significance ofinflammation in both term and preterm parturition. During the onset oflabor, there is a systemic increase in a number of pro-inflammatoryfactors including prostaglandins, cytokines, and manganese superoxidedismutase. In addition, inflammation has been strongly implicated ininfection-driven preterm labor.

Oxytocin is thought to initiate labor by exerting two distinct effects:directly inducing contraction of the uterine myometrium, and enhancingthe synthesis and release of contractile prostaglandins from the uterineendometrium/decidua. By inhibiting oxytocin signal transduction, thedirect (contractile) and indirect (enhanced prostaglandin synthesis)effects of oxytocin on the uterus may be achieved. Additionally,treatment of human decidua with oxytocin results in the stimulation ofprostaglandin F2α production. This suggests that a complimentary rolefor oxytocin signalling in uterine tissues exists, whereby oxytocin caninteract not only both directly with the myometrium in stimulatinguterine contractions, but also indirectly via the formation ofprostaglandins in other tissues.

There is recent evidence correlating the activity of the contractileprostaglandin F receptor with the onset and during the progression oflabor. Recent reports also indicate that oxytocin induces production ofprostaglandins in human myometrial cells via potentiation ofcylooxygenase 2 (COX-2). Such a mechanism may explain the sustainedrelease of prostaglandins in uterine tissue that promotes labor. Acombination therapy including a prostaglandin F2α receptor antagonist,such as compound I or a salt thereof (e.g., compound III) and anoxytocin receptor antagonist may therefore be useful for the treatmentand/or prevention or preterm labor. Additionally, the combination of anoxytocin receptor antagonist and a prostaglandin F2α receptor antagonistmay be more efficacious for treating preterm labor than currentregimens. Synergistic effects may be observed in the prevention of bothcontractile and inflammatory processes that underlie preterm labor, asthe dose(s) of an oxytocin receptor antagonist administered to a patientmay be lower when administered in combination with a prostaglandin Freceptor antagonist relative to the doses that may be administered to apatient receiving an oxytocin receptor antagonist alone.

Compound I or a salt thereof, such as compound III, can be administeredwith one or more additional agents, such as an oxytocin receptorantagonist, in order to reduce the occurrence of uterine contractionsand to delay the onset of labor. For instance, compound I or a saltthereof, such as compound III, can be administered simultaneously with,admixed with, or administered separately from an oxytocin receptorantagonist. Exemplary oxytocin receptor antagonists for use inconjunction with the compositions and methods of the invention includeatosiban, retosiban, barusiban, epelsiban, and nolasiban, or aderivative thereof. For instance, compound I or a salt thereof, such ascompound III, may be administered prior to, after, or simultaneouslywith nolasiban, or a derivative thereof, in order to delay the onset oflabor in a subject, e.g., by one or more days or weeks, such as fromabout 1 day to about 16 weeks (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 days, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,or 16 weeks).

Pharmaceutical Compositions

Compound I or a salt thereof, such as compound III, can be formulatedinto a pharmaceutical composition for administration to a subject, suchas a pregnant female human subject, in a biologically compatible formsuitable for administration in vivo. Accordingly, in one aspect, thepresent invention provides a pharmaceutical composition containingcompound I or a salt thereof, such as compound III, in admixture with asuitable diluent, carrier, or excipient. Compound I or a salt thereof,such as compound III, can be administered, for example, orally or byintravenous injection. Under ordinary conditions of storage and use, apharmaceutical composition may contain a preservative, e.g., to preventthe growth of microorganisms. Conventional procedures and ingredientsfor the selection and preparation of suitable formulations aredescribed, for example, in Remington: The Science and Practice ofPharmacy (2012, 22^(nd) ed.) and in The United States Pharmacopeia: TheNational Formulary (2015, USP 38 NF 33).

Pharmaceutical compositions may include sterile aqueous solutions,dispersions, or powders, e.g., for the extemporaneous preparation ofsterile solutions or dispersions. In all cases the form may besterilized using techniques known in the art and may be fluidized to theextent that may be easily administered to a subject in need oftreatment.

A pharmaceutical composition may be administered to a subject, e.g., ahuman subject, alone or in combination with pharmaceutically acceptablecarriers, as noted herein, the proportion of which may be determined bythe solubility and/or chemical nature of the compound, chosen route ofadministration, and standard pharmaceutical practice.

Compositions for Combination Therapy

Compound I or a salt thereof, such as compound III, can be used alone orin combination with one or more additional agents useful for theinhibition of uterine contractions and/or luteolysis, such as atosiban,retosiban, barusiban, epelsiban, and nolasiban, or a derivative thereof.Compound I or a salt thereof, such as compound III, can be admixed withan additional active agent, such as an oxytocin receptor antagonistdescribed herein, and administered to a patient in a single composition,or compound I or a salt thereof, such as compound III, can beadministered to a patient separately from an additional active agent.For instance, compound I or a salt thereof, such as compound III, and anadditional active agent can be sequentially administered to a patient.In combination treatments, the dosages of one or more of the therapeuticcompounds may be reduced from standard dosages when administered alone.For example, doses may be determined empirically from drug combinationsand permutations or may be deduced by a physician of skill in the art.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a description of how the compositions and methodsdescribed herein may be used, made, and evaluated, and are intended tobe purely exemplary of the invention and are not intended to limit thescope of what the inventors regards as their invention.

Example 1 Preparation of Compounds I and III

Compound I, and the chloride salt thereof (compound III), were preparedaccording to Scheme 1, shown below. This Example will describe each ofthe stages carried out to synthesize compound I, designated Stages 1-6.

Stage 1: Preparation of2-[1-(4-fluorophenyl)-3-hydroxypropylcarbamoyl]thiazolidine-3-carboxylicacid tert-butyl ester

To a suitably sized flask (vessel A),3-(butoxycarbonyl)-1,3-thiazolidine-(2S)-carboxylic acid (1 wt) wasadded, followed by tetrahydrofuran and the flask contents weresubsequently cooled to −35° C. to about −45° C. N-methylmorpholine (1.18vol) were then added to the flask while maintaining the temperaturebetween −30° C. and −40° C. Isobutyl chloroformate (0.58 vol) were thenadded to the flask while maintaining the temperature between −30° C. and−40° C.

To a separate vessel (vessel B),(3S)-amino-3-(4-fluorophenyl)propan-1-ol (0.76 wt) and THF were addedand the vessel was mixed thoroughly until the bulk solids dissolved.

The (3S)-amino-3-(4-fluorophenyl)propan-1-ol solution of vessel B wasthen added to the reaction vessel A while maintaining the temperaturebetween −30° C. and −40° C. The flask contents were then allowed to warmto 15° C. to 25° C. over a period of 1 h to 24 h. The reaction mixturewas stirred at 15° C. to 25° C. until the reaction was observed to becomplete. The reaction mixture was concentrated to dryness, and ethylacetate was subsequently added to the residue, followed by saturatedaqueous ammonium chloride. The organic phase was separated and washedwith saturated aqueous ammonium chloride solution. The organic phase wasthen separated and washed with saturated aqueous sodium hydrogencarbonate solution. The organic phase was then dried over sodiumsulfate, filtered, and the filtrate concentrated at 35° C. to 40° C.until the ethyl acetate content was 10% by weight (w/w) to yield2-[1-(4-fluorophenyl)-3-hydroxypropylcarbamoyl]thiazolidine-3-carboxylicacid tert-butyl ester.

Stage 2: Preparation of 3-(biphenyl-4-sulfonyl)thiazolidine-2-carboxylicacid [1-(4-fluoropphenyl)-3-hydroxypropyl]-amide

To a suitably sized flask (vessel A),2-[1-(4-fluorophenyl)-3-hydroxypropylcarbamoyl]thiazolidine-3-carboxylicacid tert-butyl ester (1 wt) was added, followed by dichloromethane. Theflask contents were subsequently cooled to −15° C. to −20° C.Hydrochloric acid (3.3 vol) was then added to the flask whilemaintaining the temperature between −15° C. and −20° C. until thereaction was observed to be complete. The reaction mixture was thencooled to −35° C. to −40° C. and tetrahydrofuran was added to themixture while maintaining the temperature between −30° C. and −40° C.N,N-diisopropylethylamine was then added to the mixture (8.16 vol) whilemaintaining the temperature between −15° C. and −45° C.4-dimethylaminopyridine (0.032 wt) was then added to the vessel whilemaintaining the temperature between −15° C. and −45° C.

In a separate vessel (vessel B), 4-biphenylsulfonyl chloride (0.85 wt)was added, followed by THF.

The 4-biphenylsulfonyl chloride solution from vessel B was added to thereaction vessel A while maintaining the temperature between −15° C. and−45° C. The contents of the reaction mixture were then allowed to warmto 15° C. to 25° C. over a period of 1 h to 24 h. Ethyl acetate wassubsequently added to the flask, followed by saturated aqueous ammoniumchloride solution. The organic phase was separated and washed withsaturated aqueous ammonium chloride solution followed by saturatedaqueous hydrogen carbonate solution. The organic phase was then driedover sodium sulfate and filtered. The filtrate was concentrated at 35°C. to 40° C. until a solid residue was obtained. Dichloromethane wasthen added to the residue and mixed at 30° C. to 35° C. Afterevaporation, ethyl acetate was then added to the residue, and the slurrywas transferred to a suitable vessel. The stirred slurry was then warmedto reflux, and then cooled to 0° C. to 5° C. The precipitated solid wascollected by filtration. The filter cake was washed ethyl acetatefollowed by tert-butyl methyl ether and the filter cake was pulled dryfor 1 h to 24 h under nitrogen to yield3-(biphenyl-4-sulfonyl)thiazolidine-2-carboxylic acid[1-(4-fluoropphenyl)-3-hydroxypropyl]-amide.

Stage 3A: Preparation of 2-tert-butoxycarbonylamino-3-methylbutyric acid3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)-3-propylester

To a suitably sized flask (vessel A), Boc-L-valine (0.48 wt),dichloromethane, and N,N-dimethylformamide were added and the mixturewas subsequently stirred under nitrogen at 15° C. to 25° C.1-hydroxybenzotriazole (HOBt, 0.3 wt) and1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl, 0.42wt) were then added to the vessel while maintaining the temperature at15° C. to 25° C. The mixture was subsequently stirred at 15° C. to 25°C. until the bulk solids dissolved in order to yield solution A.

To a separate vessel (vessel B),3-(biphenyl-4-sulfonyl)thiazolidine-2-carboxylic acid[1-(4-fluorophenyl)-3-hydroxypropyl]amide (1.0 wt), dichloromethane, andN,N-dimethylformamide were added, and the mixture was subsequentlystirred at 15° C. to 25° C. under nitrogen. 4-dimethylaminopyridine(0.27 wt) was then added to the vessel while maintaining the temperaturebetween 15° C. to 25° C. The mixture was stirred at this temperatureuntil the bulk solids dissolved (typically 5 to 15 minutes) to yieldsolution B.

Solution A was then added to solution B while maintaining thetemperature between 15° C. and 30° C. The mixture was stirred at thistemperature until the reaction was observed to be complete. The reactionmixture was concentrated to remove volatile solvents. Ethyl acetate wassubsequently added to the flask, followed by 10% w/w aqueous citric acidsolution. The aqueous phase was separated and extracted with ethylacetate. The combined organic phases were washed with a mixture of 10%w/w aqueous citric acid solution and saturated aqueous sodium chloridesolution were added, followed by saturated aqueous ammonium chloridesolution, saturated aqueous sodium hydrogen carbonate and saturatedaqueous sodium chloride solution. The organic phase was then dried overmagnesium sulfate, filtered, and the filter cake washed with ethylacetate. The filtrates were concentrated until a solid residue wasobtained to yield crude 2-tert-butoxycarbonylamino-3-methylbutyric acid3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)-3-propylester.

Stage 3B: Purification of 2-tert-butoxycarbonylamino-3-methylbutyricacid3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)-3-propylester

To purify 2-tert-butoxycarbonylamino-3-methylbutyric acid3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)-3-propylester, the crude product (1 wt) and dichloromethane were mixed in avessel until the bulk solids dissolved. The solution was then loaded onto silica followed by the addition of dichloromethane. The product waseluted with ethyl acetate:heptanes. Fractions containing the productwere combined and concentrated to dryness under vacuum at a water bathtemperature of 35° C. to 40° C. to yield purified2-tert-butoxycarbonylamino-3-methylbutyric acid3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)-3-propylester.

Stage 4: Preparation of 2-amino-3-methylbutyric acid3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)propyl ester methanesulfonate

To a suitably sized flask, 2-tert-butoxycarbonylamino-3-methylbutyricacid3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)-3-propylester (1 wt) was added, followed by 1,4-dioxane and the mixture wasstirred under nitrogen. Methanesulfonic acid (0.18 wt) was subsequentlyadded, and the flask contents were heated to 68° C. to 73° C. Thereaction was stirred at this temperature until the reaction was observedto be complete by ¹H NMR analysis. The reaction mixture was subsequentlycooled to 35° C. to 40° C. and concentrated to dryness at thistemperature. The residue was then dissolved in THF and concentrated todryness at 35° C. to 40° C. This azeo-drying cycle was repeated untilthe 1,4-dioxane content was less than 1.0% w/w to yield2-amino-3-methylbutyric acid3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)propyl ester methanesulfonate.

Stage 5: Preparation of 2-amino-3-methylbutyric acid3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)propyl ester (Compound I)

To a suitably sized flask, 2-amino-3-methylbutyric acid3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)propyl ester methanesulfonate (1 wt) was added, followed bydichloromethane. The flask contents were subsequently cooled to 5° C. to15° C. Aqueous sodium hydrogen carbonate solution was added to themixture while maintaining the temperature between 5° C. and 25° C. Thephases were subsequently separated, and the organic phase was re-addedto the vessel, followed by saturated aqueous sodium hydrogen carbonatesolution while maintaining the temperature at 5° C. to 25° C. Theaqueous and organic layers were then separated, and the organic phasewas dried over magnesium sulfate, filtered, and the filter cake washedwith dichloromethane. The combined organic layers were then concentratedto dryness at 40° C. to 45° C. until the dichloromethane content was 2%w/w to yield 2-amino-3-methylbutyric acid3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)propyl ester (compound I).

Stage 6: Preparation of 2-amino-3-methylbutyric acid3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)propyl ester hydrochloride (Compound III)

To a suitably sized flask, water (1.66 vol) was added, followed byhydrochloric acid (0.18 vol), and the temperature of the mixture wasadjusted to 15° C. to 25° C. The solution was then filtered, and thefiltered solution was added to a suitably sized flask (vessel A)followed by ethanol and ethyl acetate. The resulting mixture was stirredunder nitrogen at 15° C. to 25° C. for at least 5 minutes.

In a suitably sized vessel (vessel B), 2-amino-3-methylbutyric acid3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)propyl ester (1 wt) was added, followed by ethanol. The contents of theflask were subsequently mixed to dissolve the bulk solids and clarifythe solution.

The solution of vessel B was then added to vessel A while maintainingthe temperature at 15° C. to 25° C. The stirred mixture was cooled to 0°C. to 5° C. and stirred at this temperature for 50 to 70 minutes. Thesolid was collected by filtration and the filter cake pulled dry undernitrogen for at least 12 hours to yield crude 2-amino-3-methylbutyricacid3-{[3-(biphenyl-4-sulfonyl)thiazolidine-2-carbonyl]amino}-3-(4-fluorophenyl)propyl ester hydrochloride.

Example 2 Pharmacodynamic Properties of Compound I and Salts ThereofNon-Clinical Pharmacology

Compound I and salts thereof are rapidly converted to compound IIfollowing gastrointestinal tract administration. Compound II is acompetitive and reversible prostaglandin F2α receptor antagonist (humanFP2α receptor K_(i)=6 nM) that is under development for the managementof pre-term labor by inhibition of premature uterine contractions.Efficacy pharmacology (tocolytic effect) has been demonstrated in amodel of spontaneous uterine activity in late-term pregnant rats.

In Vitro Pharmacology

The potency of inhibition of compound I and compound II on prostaglandinF2α receptor was assessed by analyzing the affinity of these compoundsfor recombinant FP receptor expressed in HEK293-EBNA cells. The resultsshow high binding affinity of compound I and compound II to the humanreceptor (see Table 1).

Selectivity of compound II was tested against all eight prostaglandinreceptor subtypes. Selectivity was approximately 10-fold versusprostaglandin E receptor 2 (EP2) and higher than 100-fold against otherreceptors. Testing the effect of 1 μM compound II against a panel of 50receptors, channels and enzymes binding sites showed high selectivityfor FP.

The functional characterization of compound II on human FP was performedin transfected HEK293-EBNA cells. Compound II was able todose-dependently inhibit the synthesis of IP3 with IC₅₀ value 60 nM.When added alone to FP/HEK293-EBNA cells, compound II tested up to 10 μMdid not induce any synthesis of IP3, indicating that the compound isdevoid of agonist activity.

In Vivo Pharmacology

The tocolytic effects of compound I and compound II were investigated ina model of spontaneous uterine activity in late-term (19-21 days ofgestation) anaesthetized pregnant rat (Kawarabayashi et al. Am. J.Obstet. Gynecol. 175:1348-1355 (1996) and Shinkai et al. J. Pharm.Pharmacol. 52:1417-1423 (2000)). Briefly, late-term pregnant female ratswere anaesthetized with urethane. One pregnant uterine horn was exposedand a polyethylene catheter bearing on the tip a latex balloon filledwith saline was inserted into the lumen. The catheter was connected toan amplifying/recording system via a pressure-transducer. Increasingdoses of compound I (as mesylate salt) or compound II were orallyadministered or injected by a 10-min i.v. infusion. For the i.v.administration the uterine contractile activity was quantified bycalculating the AUC during the 10 min injection period.

The percent variation of the AUC values relative to the spontaneousuterine response observed after each compound administration wascalculated in comparison to the value recorded before the firstdose-administration (basal value). The effect of compound I or compoundII was evaluated by comparing pre- and post-treatment luminal uterinepressure values. For the oral administration the same computationprocedure was applied at different time points after treatment.Statistical differences between treatment groups at each time-point weredetermined by using one-way ANOVA followed by Tukey test. Both compoundsintravenously or orally administered were able to markedly reducespontaneous uterine contractions by around 40-50% (maximal effectobtained at 30 mg/kg by i.v. route and 60 mg/kg by oral route). Theintravenous activity was comparable or slightly higher than that of thetocolytic drug atosiban licensed in the European Union.

The inhibitory effect following the oral administration appeared with afast onset (5-15 min after administration) and remained at sustainedlevel up to the end of the observation period of 3 h. (FIG. 3)

By single oral dose, significant inhibition of uterine contractions areachieved at 30 mg/kg.

In vitro pharmacology studies thus showed the high affinity of compoundI and compound II for the human FP receptor. When administered by theintravenous or oral route, these compounds were able to markedly reducespontaneous uterine contractions by around 40-50% when investigated in amodel of spontaneous uterine activity in late-term (19-21 days ofgestation) anaesthetized pregnant rats.

Example 3 Crystal Screens of Compound I Salts

This example describes experiments conducted to generate andcharacterize crystalline salt forms of compound I.

Summary

The mesylate salt of compound I was determined to be amorphous by XRPD.Attempts to crystallize the material were not successful. The free basewas synthesized from the mesylate salt and was used in the preparationof a variety of salts. A crystalline hydrosulfate salt was obtaineddirectly from the salt synthesis. Three salts were crystallized usingdifferent solvent mixtures and crystallization techniques:hydrochloride, fumarate and dihydrophosphate. The hydrochloride saltappeared to exhibit low hygroscopicity, extended stability at elevatedrelative humidity (RH), and assumes a single crystal form whencrystallized from a variety of distinct experimental conditions.

The crystalline HCl salt was obtained in two evaporation experiments anda slurry experiment. The same XRPD pattern was observed in each case.Based on thermal data, the material had some residual solvent; aprobable melting point was approximately 146-147° C. Partialdecomposition likely occurred during the melt. The hydrochloride saltwas non-hygroscopic based on moisture balance data.

The crystalline hydrosulfate salt was likely solvated and decomposedabove approximately 100° C. The material was stable at relativehumidities up to approximately 65%.

The crystalline dihydrophosphate and fumarate salt were hygroscopic atapproximately 65% RH. Attempts to scale up the salts were not successfuldue to high laboratory humidity. Thus, only partial characterization wasavailable for these salts.

The hydrochloride, hydrosulfate, and fumarate salt showed comparableaqueous solubilities (below 1 mg/mL, see FIG. 8).

Experimental

X-ray powder diffraction analyses described herein were carried out on aShimadzu XRD-6000 X-ray powder diffractometer using Cu Kα radiation. Theinstrument is equipped with a long fine focus X-ray tube. The tubevoltage and amperage were set at 40 kV and 40 mA, respectively. Thedivergence and scattering slits were set at 1° and the receiving slitwas set at 0.15 mm. Diffracted radiation was detected by a NaIscintillation detector. A theta-two theta continuous scan at 3°/min (0.4sec/0.02° step) from 2.5 to 40° 2θ was used. A silicon standard wasanalyzed each day to check the instrument alignment. Samples wereanalyzed with a silicon sample holder.

X-ray powder diffraction analyses described herein were also performedon an Inel XRG-3000 diffractometer, equipped with a curvedposition-sensitive detector with a 2θ range of 120°. Real time data wascollected using Cu Kα radiation starting at approximately 4° 2θ at aresolution of 0.03° 2θ. The tube voltage and amperage were set to 40 kVand 30 mA, respectively. The monochromator slit was set at 5 mm by 160μm. Patterns are displayed from 2.5 to 40° 2θ. Samples were prepared foranalysis by packing them into thin-walled glass capillaries. Eachcapillary was mounted onto a goniometer head that is motorized to permitspinning of the capillary during data acquisition. The samples wereanalyzed for 5 or 10 min. Instrument calibration was performed dailyusing a silicon reference standard.

The DSC analyses described herein were carried out on a TA Instrumentsdifferential scanning calorimeter 2920. The instrument was calibratedusing indium as the reference material. Samples were placed into astandard aluminum DSC pan, the pan was crimped, and the weightaccurately recorded. The samples were equilibrated at 25° C. and heatedunder a nitrogen purge at a rate of 10° C./min up to 350° C. Indiummetal was used as calibration standard.

The TG analyses described herein were carried out on a TA Instruments2950 thermogravimetric analyzer. The calibration standards were nickeland ALUMEL™. Samples were placed in an aluminum sample pan and insertedinto the TG furnace. The samples were first equilibrated at 25° C., thenheated under a stream of nitrogen at a heating rate of 10° C./min up to350° C.

The solution ¹H nuclear magnetic resonance (NMR) spectra describedherein were acquired at ambient temperature with a Varian UNITYINOVA-400spectrometer at a ¹H Larmor frequency of 399.8 MHz. Samples weredissolved in methanol-d4, methylene chloride-d2, or chloroform-d3. Thespectra were acquired with a ¹H pulse width of 7.8 or 8.6 μs, a 2.50second acquisition time, a 5 second delay between scans, a spectralwidth of 4095 or 6400 Hz with 20474 or 32000 data points, and 16 or 40co-added scans. The free induction decay (FID) was processed using theVarian VNMR 6.1C software with 65536 points and an exponential linebroadening factor of 0.2 Hz to improve the signal-to-noise ratio. Thespectra were referenced to internal tetramethylsilane (TMS) at 0.0 ppmor the residual solvent peak.

The FT-Raman spectra described herein were acquired on a FT-Raman 960 or860 spectrometer (Thermo Nicolet). This spectrometer uses an excitationwavelength of 1064 nm. Approximately 0.5-0.7 W of Nd:YVO4 laser powerwas used to irradiate the samples. The Raman spectra were measured withan indium gallium arsenide (InGaAs) detector. The samples were preparedfor analysis by placing the material in a glass capillary and then intoa gold-coated capillary holder in the accessory. A total of 256 samplescans were collected from 3600 to 100 cm-1 at a spectral resolution of 4cm-1, using Happ-Genzel apodization. Wavelength calibration wasperformed using sulfur and cyclohexane.

Moisture sorption/desorption (MB) data were collected on a VTI SGA-100Vapor Sorption Analyzer. Sorption and desorption data were collectedover a range of 5% to 95% relative humidity (RH) at 10% RH intervalsunder a nitrogen purge. Samples were not dried prior to analysis.Equilibrium criteria used for analysis were less than 0.0100% weightchange in 5 minutes, with a maximum equilibration time of 3 hours if theweight criterion was not met. Data were not corrected for the initialmoisture content of the samples. NaCl and PVP were used as calibrationstandards.

Preparation of Compound I

Multiple attempts were made to generate the free base of compound I fromthe mesylate salt, the results of which are described in FIG. 4.Initially, one equivalent of sodium hydroxide was used per equivalent ofthe salt. Proton NMR indicated presence of methanesulfonic acid peaks. Acomplete reaction was achieved when the mesylate salt in methylenechloride and a NaOH solution in water were mixed at a 1:2 salt: baseratio. The organic layer was separated after several washes andevaporated. The resulting paste-like or viscous oily material was driedin vacuum to yield an amorphous solid. The free base was analyzed by ¹HNMR and Raman spectroscopy (FIG. 15 and FIG. 16, respectively).Subsequent salt screen studies used the free base as the startingmaterial (summarized in FIGS. 5-7).

Salt Screen of Compound I

Twelve salts of compound I were prepared. A crystalline hydrosulfatesalt was precipitated by addition of approximately 25 molar excess ofsulfuric acid to a free base solution in acetone. The other salts fromthe synthesis step appeared to be non-birefringent by microscopy oramorphous by XRPD (FIGS. 5-7). The benzenesulfonate, citrate,ethanesulfonate, hydrochloride, hydrosulfate and sulfate salts wereanalyzed by proton NMR.

Crystallization experiments on the compound I salts are summarized inFIGS. 5-7. The following salts were crystallized: hydrochloride,fumarate, and dihydrophosphate.

The chloride salt was crystallized from a 1:1 mixture of acetone:toluene, a mixture of methylene chloride: ethyl ether, and an acetoneslurry. The same XRPD pattern was observed in all the experiments andwas designated as form A (FIG. 7). The crystalline fumarate salt wasobtained from slow evaporation of a 1:1 methanol: toluene solution. TheX-ray pattern was designated as pattern B. The hydrosulfate anddihydrophosphate salt exhibited very similar XRPD patterns (designatedas pattern X). The counterions HSO₄ ⁻ and H₂PO₄ ⁻ are similar in sizeand small compared to the free base molecule, therefore, similar crystalstructures are likely for the hydrosulfate and dihydrophosphate salt.Attempts to crystallize the mesylate salt yielded viscous or glassysolid materials.

Characterization of the Free Base and Mesylate Salt of Compound I

The proton NMR spectrum of the free base showed two doublets atapproximately 1 ppm corresponding to the methyl groups of the valinefragment. The methyl groups are at the chiral carbon center and,therefore, are not equivalent in proton NMR. Two doublets for the methylgroups were observed for the following compound I salts: besylate,citrate, esylate, hydrosulfate (more overlapped) and sulfate (moreoverlapped). In the ¹H NMR spectra of the mesylate salt and the chloridesalt, the doublet at ˜1 ppm corresponding to six hydrogen atoms resultedfrom a complete overlap of two doublets of the methyl groups (FIG. 13and FIG. 21).

A homonuclear decoupling ¹H NMR experiment on the free base confirmedthe methyne (CH) hydrogen multiplet at approximately 2 ppm (FIG. 18). A¹H NMR spectrum of the free base recorded in the absence ofpre-irradiation of either methyl group is shown at the bottom of FIG.18. Irradiation of each methyl group (top, middle) resulted in asimplified methyne multiplet with the same number of lines (5). If thetwo doublets corresponded to different diastereoisomers, two types ofmultiplets, the original and the simplified, would be observed.

Characterization of the Chloride Salt of Compound I (Compound III)

The crystalline chloride salt was analyzed by thermal techniques, ¹H NMRand automated moisture sorption/desorption analysis. The endotherm atapproximately 147° C. in DSC appeared broader than what is typicallyobserved for the melting endotherm. A weight loss of approximately 4%was observed from 25 to 160° C. (acetone slurry sample analyzed, FIG.20). The ¹H NMR of the chloride salt was consistent with the structure(FIG. 21). However, the data cannot be correlated with the weight lossin the thermal analyses because a different sample was analyzed (slowevaporation of a 1:1 acetone: toluene mixture). The chloride salt froman acetone slurry was vacuum-dried at approximately 50° C. for 1 day.The resulting sample was similar to the original salt by XRPD (FIG. 22).The thermal data are presented in FIG. 23. Based on comparison of thethermal data, the dried material had lower weight losses between 25 and100° C. (0.2% vs. 0.6% for the original chloride salt) and 100 and 160°C. (2.5% vs. 3.5%) (FIG. 24). This indicated that some solvent had beenremoved on vacuum drying. However, the endotherm at approximately146-147° C. in DSC was still broad (FIG. 25). Partial decompositionprobably occurred during the melt (note the degrading baseline and thecorresponding weight loss in TG).

The chloride salt of compound I did not deliquesce after 2 days atapproximately 95% RH. Moisture sorption/desorption data are summarizedin FIG. 27 and displayed in FIGS. 26 and 28. Minimal weight loss wasobserved on equilibration at 5% RH. Approximately 0.9% weight gainoccurred on sorption from 5 to 95% relative humidity. The sampledisplayed approximately 0.7% weight loss upon desorption. XRPD analysison the post-MB sample exhibited an X-ray pattern similar to that for thestarting material (FIG. 29).

Characterization of the Hydrosulfate and Sulfate Salts of Compound I

Both the hydrosulfate and sulfate salt of compound I were prepared. Thehydrosulfate salt was precipitated from an acetone solution of the freebase by addition of approximately 25 molar excess of sulfuric acid. Theprecipitate was found to be crystalline by XRPD (FIG. 38). Thermal datafor the hydrosulfate salt are given in FIG. 32. A broad endotherm atapproximately 68° C. corresponded to a weight loss of approximately 1%and was likely due to desolvation (dehydration). Decomposition occurredat higher temperatures. It did not deliquesce after 3 days atapproximately 65% RH (FIG. 32). The sulfate salt was prepared using twoequivalents of the free base per one equivalent of the acid. Attempts tocrystallize the sulfate salt of compound I were not successful (FIGS.5-7). The hydrosulfate and the sulfate salt were analyzed by proton NMR(FIG. 33 and FIG. 34). Differences were noted in the NMR spectra. Forexample, the methyl groups of the valine fragment appeared to havedifferent coupling.

Characterization of the Dihydrophosphate Salt of Compound I

The dihydrophosphate salt was crystallized from a 1:1 methyl ethylketone: n-butyl acetate mixture (FIGS. 5-7). It exhibited an X-raypattern similar to that of the hydrosulfate salt (FIG. 43).Characterization of the dihydrophosphate salt was limited to XRPD due tosample loss during the analysis. Attempts to prepare additionalquantities of the crystalline salt were not successful. A lowcrystalline material was generated during the first attempt (FIGS. 5-7).A recrystallization of the low crystalline salt yielded a viscous solid.The material remained viscous after it had been dried in vacuum. Thelaboratory humidity was approximately 62% RH during the scale-upcrystallization and likely affected the material due to itshygroscopicity. No further attempts to crystallize the dihydrophosphatesalt were undertaken.

Characterization of the Fumarate Salt of Compound I

A small amount of the fumarate salt was crystallized from a methanol:toluene 1:1 mixture (FIGS. 5-7). Attempts to scale up the crystallinesalt were carried out at the laboratory humidity of approximately 62% RHand were not successful. Mostly oily materials resulted, although somecrystalline solid was present by microscopy. Drying the viscous solid invacuum yielded mostly amorphous material. The originally preparedcrystalline salt was used for seeding experiments. However, nocrystalline materials were generated. The hygroscopic nature of thefumarate salt was confirmed in relative humidity studies.

The fumarate salt appeared to be moisture sensitive. The crystallinesalt was stable at approximately 43 and 53% relative humidities, andbegan to deliquesce within the first day at approximately 65% RH. Yellowoil formed after 3 days at 65% RH (approximately 4% of moisture gained).

Conclusions

The mesylate salt of compound I was found to be amorphous by XRPD.Attempts to crystallize the material were not successful.

The free base of compound I was synthesized from the mesylate salt andused in preparation of 12 salts. A crystalline hydrosulfate salt wasobtained directly from the salt synthesis. Three salts were crystallizedusing different solvent mixtures and crystallization techniques:hydrochloride, fumarate and dihydrophosphate. The chloride salt appearedto be the best candidate for further development. The crystallinehydrosulfate salt was likely solvated and decomposed above approximately100° C. The material was stable at relative humidities up toapproximately 65%. The crystalline HCl salt was obtained in twoevaporation experiments and a slurry experiment. The same XRPD patternwas observed. Based on thermal data, the material had some residualsolvent; a probable melting point was approximately 146-147° C. Partialdecomposition likely occurred during the melt. The chloride salt wasnon-hygroscopic based on moisture balance data. The crystallinedihydrophosphate and fumarate salt were hydroscopic at approximately 65%RH. Attempts to scale up the salts were not successful due to highlaboratory humidity. Thus, only partial characterization was availablefor these salts.

Example 4 Monitoring Caco-2 Cell Permeability of the Mesylate Salt ofCompound I

The bioavailability of orally administered drugs depends to a greatextent on the capability of being transported across the intestinalbarriers. Caco-2 cells, derived from a human colon adenocarcinoma,established by J. Fogh for its ability to achieve a higher degree ofenterocytic differentiation, can be used as an in vitro model for theinvestigation of transport of drugs through the intestinal epithelium.These cells form a monolayer of polarized epithelial cells when grownonto collagen-coated polycarbonate membrane. The monolayer ofdifferentiated cells represents a relevant model for the smallintestinal epithelium. The process of differentiation starting at cellconfluence leads to the formation of a brush border with well-developedmicrovilli, tight apical junctions, and a polarized distribution ofmembrane components, including enzymes, receptors, transport systems,ion channels and lipid molecules

The purpose of the study was in a first step to assess the non-specificbinding of compound I in the Caco-2 cell test system (without cells)and, in a second step, to assess the conversion of compound I intocompound II and to determine if the transport of compound I acrossCaco-2 cell monolayers is mediated by the PepT1 transporter protein.

Materials

Caco-2 cell line (human colon adenocarcinoma cells) was obtained fromcontrolled cell Banks (Biosearch S.p.A, Gerenzano-Italy). Dulbecco'smodified Eagles's Medium (DMEM), Fetal Bovine Serum, Non essential aminoacids solution, L-Glutamine 200 mM, ennicillin/Streptomycin Solution,Trypsin-EDTA solution without Calcium and Magnesium were purchased fromCelbio (Milan, Italy). HEPES, Hank's Balanced Salt Solution (HBSS),Dulbecco's Phosphate Buffered Saline (PBS), Dimethyl Sulphoxide (DMSO),Glycine-Sarcosine (Gly-Sar) were purchased from Sigma (Milan, Italy).

Experimental

The Caco-2 cells were cultured in DMEM supplemented with 10% FetalBovine Serum, 2% L-Glutamine 200 mM and 1% non-essential amino acidssolution.

The cells were stored frozen in cryotubes under liquid nitrogen, as 1 mLvolumes of cell suspension in Fetal Bovine Serum containing 10% DMSO.Cells used for the experiments will be kept in culture for no longerthan one month.

When necessary, frozen vials of Caco-2 cells were rapidly thawed at 37°C. in a water bath by gently swirling up to semi-complete thawing. Thenthe cell suspension was added drop by drop to 10 mL of culture medium.The cell suspension was then centrifuged for 7 minutes at 900-1000 rpm,the supernatant was removed and the cell pellet reconstituted in themedium and distributed into 75 cm² flasks containing medium. The flaskswere incubated at 37° C. in an atmosphere of 5% CO₂. The cells wereserially subcultured when near-confluent monolayers were obtained. Themedium of each flask was removed and the monolayer was washed with 10-15mL of Dulbecco's Phosphate Buffer Saline (PBS).

Trypsin-EDTA solution was added to the cell monolayer, incubated at 37°C. and tapped gently at intervals to dislodge the cells. Completedetachment and disaggregation of the cell monolayer was confirmed bymicroscopy examination. The cells were then re-suspended in 10 mL ofcomplete medium and centrifuged for 7 minutes at 900-1000 rpm. Thesupernatant was discarded; the cells were resuspended in culture mediumand plated at 2.5×105 cell/mL in 175 cm2 flasks.

The cells from flasks of near-confluent cultures were detached anddisaggregated by treatment with trypsin as described above. The cellswere resuspended in culture medium and counted. The cell suspension wasdiluted with medium to give about 1×10⁶ cells/mL and 300 μL of cellsuspension was put onto the apical compartment of each Transwell (6.5 mmdiameter, 0.4 μm pore size). 600 μL of culture medium were put into thebasolateral compartment. The plates were incubated at 37° C. in ahumidified atmosphere of 5% CO2 in air for 15-21 days, changing themedium every 48-72 hours.

The integrity of each Caco-2 cell monolayer was evaluated byTransepithelial Electrical resistance (TEER), both pre-experiment and atthe end of the incubation time. TEER, expressed as ohms×cm², wasmeasured in the Transwells using the Millicell-ERS (Millipore). Themonolayer is considered well differentiated when TEER value is higherthan 800 ohms×cm².

The integrity of each Caco-2 cell monolayer was evaluated at the end ofthe incubation time by Lucifer Yellow. Post experiment the Transwellswere washed twice with transport buffer. 200 μL of Lucifer Yellow at theconcentration of 100 μM in HBSS were distributed in the apicalcompartment, while 400 μL of HBSS were added to the basolateralcompartment. The transwells were incubated at 37° C. for 1 hour. Theamount of Lucifer Yellow was quantitated in the basolateral compartmentat 535 nm wavelength against a standard Lucifer Yellow curve in the samesaline solution, using a Microplate Spectrofluorometer (EG & G WALLAC).The monolayer is considered not damaged if <1% Lucifer Yellow isdetected in the basolateral compartment.

Assessment of Non-Specific Binding to Cell-Free Transwells

Non-specific binding and recovery was assessed across cell-freetranswells.

Compound I was tested at 1.5, 3 and 6 μM in duplicate cell-freetranswells. The test was performed in a pH gradient between the apicaland the basolateral compartment. The apical compartment (donor) had abuffer pH of 6.5 while the basolateral compartment (receiver) had abuffer pH of 7.4. The following sampling times were performed: 60 and120 min for the basolateral compartment (receiver) and 120 min for theapical compartment (donor). Samples obtained were analyzed by LC-MS,both compound I and compound II were monitored in order to assesspercent of recovery.

Assessment of Stability of Compound I and Compound II

Stability of both compound I and compound II was assessed during thetest. These compounds were dissolved in HBSS buffer (1% DMSO finalconcentration) at the concentrations of 1.5, 3 and 6 μM. An aliquot ofeach solution was sampled at time zero (t=0) to assess the startingconcentrations of the compounds. The solutions were incubated at 37° C.for the duration of the transport experiment. An aliquot of eachsolution was sampled at the end of experiment (t=120) to assess thefinal concentrations of compound I and compound II. Samples wereanalyzed by LC-MS.

Assessment of Bidirectional Permeability of Compound I

Compound I was dissolved in HBSS buffer (1% DMSO final concentration) atthe concentrations of 1.5, 3 and 6 μM. Each concentration/sampling timewas run in duplicate well. The test was performed in a gradient pH: theapical compartment (mucosal) was at pH 6.5, the basolateral compartment(serosal) was at pH 7.4.

Apical to basolateral (A→B, mucosal to serosal) transport: 200 μL ofeach concentration of compound I was added to the apical compartment and400 μL of HBSS was added to the basolateral compartment. The plates wereincubated at 37° C. An aliquot of the basolateral compartment wassampled after 60 and 120 min (t=60 and t=120). An aliquot of the apicalcompartment was sampled at the starting time (t=0) and after 120 min.(t=120).

Basolateral to apical (B→A, serosal to mucosal) transport: 400 μL ofeach concentration of compound I was added to the basolateralcompartment and 200 μL of HBSS was added to the apical compartment. Theplates were incubated at 37° C. An aliquot of the apical compartment wassampled after 60 and 120 min (t=60 and t=120). An aliquot of thebasolateral compartment was sampled at the starting time (t=0) and after120 min. (t=120). All samples were analyzed by LC/MS monitoring bothcompound I and the appearance of compound II.

Assessment of Bidirectional Permeability of Compound II

Compound II was dissolved in HBSS buffer (1% DMSO final concentration)at the concentrations of 1.5, 3 and 6 μM. Each concentration/samplingtime was run in duplicate well. The test was performed in a gradient pH:the apical compartment (mucosal) was at pH 6.5, the basolateralcompartment (serosal) was at pH 7.4.

Apical to basolateral (A→B, mucosal to serosal) transport: 200 μL ofeach concentration of compound II was added to the apical compartmentand 400 μL of HBSS was added to the basolateral compartment. The plateswere incubated at 37° C. An aliquot of the basolateral compartment wassampled after 60 and 120 min (t=60 and t=120). An aliquot of the apicalcompartment was sampled at the starting time (t=0) and after 120 min.(t=120).

Basolateral to apical (B→A, serosal to mucosal) transport: 400 μL ofeach concentration of compound II was added to the basolateralcompartment and 200 μL of HBSS was added to the apical compartment. Theplates were incubated at 37° C. An aliquot of the apical compartment wassampled after 60 and 120 min (t=60 and t=120). An aliquot of thebasolateral compartment was sampled at the starting time (t=0) and after120 min. (t=120). All samples were analyzed by LC/MS monitoring compoundII.

Inhibition of Mucosal-to-Serosal Transport of Compound I by PepT1Substrate (Gly-Sar)

The differentiated cells were pre-treated for 30 min. with 10 mM ofGly-Sar in order to block the active transporter PepT1.

Compound I was dissolved in HBSS buffer (1% DMSO final concentration) atthe concentrations of 1.5, 3 and 6 μM. Each concentration/sampling timewas run in duplicate well. The test was performed in a gradient pH: theapical compartment (mucosal) was at pH 6.5, the basolateral compartment(serosal) was at pH 7.4.

Apical to basolateral (A→B, mucosal to serosal) transport: 200 μL ofeach concentration of compound I was added to the apical compartment and400 μL of HBSS was added to the basolateral compartment. The plates wereincubated at 37° C. An aliquot of the basolateral compartment wassampled after 60 and 120 min (t=60 and t=120). An aliquot of the apicalcompartment was sampled at the starting time (t=0) and after 120 min.(t=120). All samples were analyzed by LC/MS monitoring both compound Iand the appearance of compound II.

Analytical Determinations

The concentrations of compound II and compound I in the post-incubationsamples were determined by a high performance liquid chromatography/massspectrometry (LC/MS) method reported in Appendices (section 7.1) withoutany further dilution.

Results

Pre-experiments TEER values of the Caco-2 cell monolayers used rangedfrom 850 to 1160Ω×cm², indicating confluent monolayer with tightjunctions. At the end of the experiments TEER values decreased inaverage of 170Ω×cm² (from 680 to 990Ω×cm²) with no influence of cellmonolayer integrity. The Lucifer Yellow test confirmed the integrity ofall monolayers post-experiments, in fact the amount of Lucifer Yellowdetected in the basolateral compartments post-experiments was always <1%in all wells. FIG. 55 reports data obtained in the non-specific bindingtest on compound I. In the test conditions compound I proved to berecovered in the apical compartment at all the doses tested. Compound Iwas not detected in the basolateral compartment at any dose tested.Non-specific binding of compound I was excluded. Compound II was notdetected in any compartment. FIG. 55 reports data obtained in thestability test on compound I and compound II. Both compounds proved tobe stable in the test conditions: HBSS buffer (2% DMSO finalconcentration) at 37° C. for 60 and 120 minutes. FIGS. 56a-56e reportdata obtained in the bi-directional permeability test on compound I.This compound did not pass through the cell monolayer. In the apical tobasolateral test compound I was not detected in the receivingcompartment after both 60 and 120 minutes, while increasingconcentrations of compound II were detected at the end of the experimentin basolateral compartment. The percentage of passage of compound II isreported in the table. At the end of the apical to basolateralexperiment, in the apical compartment low recovery of compound I wasobserved, while increased concentrations of compound II were detected(high recovery). The increased concentration of compound II after 120min in the apical compartment could be explained by the presence ofextra- and intracellular esterases in the Caco-2 cell able tode-esterify compounds (Kern et al. J. Agric. Food Chem. 51: 7884-7891(2003)). In the basolateral to apical test compound I was not detectedin the receiving compartment, while low concentrations of compound IIwere detected. Therefore, compound I is likely transferred andtransported as compound II through the Caco-2 monolayer. FIGS. 57a-57ereport data obtained in the bi-directional permeability test on compoundII. This compound showed a good percentage of passage apical tobasolateral and a low rate of permeability from basolateral to apicalcompartment. Papp was calculated because concentration in the donorcompartments was known. Compound II has a good passive passage throughthe Caco-2 monolayer. No efflux was detected. FIGS. 58a-58c report dataobtained in the inhibition test, in which the Caco-2 cell monolayer waspre-treated with 10 mM Gly-Sar (in order to saturate PepT1 transporter).Compound I was not detected in the receiving compartment, while apassage of compound II was observed. The percentage of passage was notlinear in this test.

Discussion

In this study the non-specific binding of compound I in the Caco-2 celltest system (without cells) was evaluated and excluded. Compound I wasstable in the test conditions. The conversion of compound I intocompound II was evaluated and confirmed in the bi-directionalpermeability test. Compound I did not pass through the cell monolayerunder the conditions tested. Compound I is therefore likely transferredand transported as de-esterfied compound II through the Caco-2 cellmonolayer.

In the bi-directional permeability test compound II showed a goodpassive passage through the Caco-2 cell monolayer. Evidence was notfound that compound II might be a substrate for an efflux transporter.

The test with Gly-Sar pre-treatment (in order to saturate PepT1transporter) showed no passage of compound I and a rate of passage ofcompound II. The transport of compound I across Caco-2 cell monolayersis likely not mediated by PepT1.

OTHER EMBODIMENTS

All publications, patents, and patent applications mentioned in thisspecification are incorporated herein by reference to the same extent asif each independent publication or patent application was specificallyand individually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from theinvention that come within known or customary practice within the art towhich the invention pertains and may be applied to the essentialfeatures hereinbefore set forth, and follows in the scope of the claims.

Other embodiments are within the claims.

The invention claimed is:
 1. A compound represented by formula (III)


2. The compound of claim 1, wherein said compound is in a crystallinestate.
 3. The compound of claim 2, wherein said compound exhibitscharacteristic X-ray powder diffraction peaks at about 7.0° 2θ, about8.1° 2θ, about 10.0° 2θ, about 12.0° 2θ, about 13.1° 2θ, about 14.1° 2θ,about 16.4° 2θ, about 18.4° 2θ, about 20.1° 2θ, about 21.0° 2θ, about23.5° 2θ, and about 29.5° 2θ.
 4. The compound of claim 3, wherein saidcompound is characterized by an X-ray powder diffraction spectrumsubstantially as depicted in FIG.
 49. 5. The compound of claim 2,wherein said compound exhibits ¹H nuclear magnetic resonance (NMR) peakscentered at about 1.1 ppm, about 3.3 ppm, about 4.9 ppm, about 5.4 ppm,about 7.1 ppm, about 7.7 ppm, about 7.9 ppm, and about 8.0 ppm.
 6. Thecompound of claim 5, wherein said compound is characterized by a ¹H NMRspectrum substantially as depicted in FIG.
 21. 7. The compound of claim2, wherein said compound exhibits an endotherm at from about 145° C. toabout 147° C. as measured by differential scanning calorimetry.
 8. Thecompound of claim 7, wherein said compound is characterized by adifferential scanning calorimetry curve substantially as depicted inFIG. 20 or FIG.
 23. 9. The compound of claim 2, wherein said compoundexhibits a weight loss of from about 0.2% to about 0.6% when heated from25° C. to 100° C. as measured by thermogravimetric analysis.
 10. Thecompound of claim 9, wherein said compound exhibits a thermogravimetricanalysis curve substantially as depicted in FIG.
 24. 11. Apharmaceutical composition comprising the compound of claim 1 and apharmaceutically acceptable excipient.
 12. The pharmaceuticalcomposition of claim 11, wherein said pharmaceutical composition is aliquid suspension.
 13. A method of synthesizing a compound representedby formula (III),

said method comprising reacting a precursor represented by formula (IV)

with a precursor represented by formula (V)

to form an amino ester, wherein X is a protecting group, and whereinsaid method further comprises reacting said amino ester with a reagentcapable of deprotecting said amino ester.
 14. The method of claim 13,wherein said protecting group is tert-butoxycarbonyl and said reagent ismethanesulfonic acid.
 15. The method of claim 13, said method comprisingreacting said precursor represented by formula (IV) with said precursorrepresented by formula (V),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-hydroxybenzotriazole,and N,N-dimethylaminopyridine.
 16. The method of claim 13, said methodcomprising synthesizing said precursor represented by formula (IV) byreacting a precursor represented by formula (VI)

with a precursor represented by formula (VII)


17. The method of claim 16, said method comprising reacting saidprecursor represented by formula (VI) with said precursor represented byformula (VII) and diisopropylethylamine.
 18. A method of making acompound represented by formula (III) in a crystalline state,

said method comprising mixing a compound represented by formula (I)

with aqueous hydrochloric acid.
 19. The method of claim 18, said methodcomprising: a) dissolving said compound represented by formula (I) inethanol; b) mixing said aqueous hydrochloric acid with ethanol and ethylacetate; c) adding said compound represented by formula (I) to saidaqueous hydrochloric acid over a period of from about 20 to about 30minutes to form a mixture and maintaining the temperature of saidmixture at from about 15° C. to about 25° C. during said adding; d)reducing the temperature of said mixture to about 5° C. following saidadding; and e) stirring said mixture for from about 50 to about 70minutes at from about 0° C. to about 5° C. following said reducing. 20.The method of claim 19, wherein said compound represented by formula (I)and said aqueous hydrochloric acid are mixed in equimolar amounts.
 21. Amethod of treating preterm labor in a human subject, said methodcomprising administering to said human subject a therapeuticallyeffective amount of a compound of formula (III)


22. The method of claim 21, wherein said human subject is characterizedby a gestational age of from about 24 to about 34 weeks.
 23. The methodof claim 21, wherein the human subject exhibits a reduction in theamplitude of uterine contractions following said administering.
 24. Themethod of claim 21, said method comprising orally administering saidcompound to said subject.